CN116949169B - Application of SMEK1 in diagnosis and treatment of cerebral arterial thrombosis - Google Patents
Application of SMEK1 in diagnosis and treatment of cerebral arterial thrombosis Download PDFInfo
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Abstract
The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to application of SMEK1 in diagnosis and treatment of ischemic cerebral apoplexy. The invention reports that SMEK1 presents fluctuation change after ischemic cerebral apoplexy for the first time and presents descending trend in a quite long time. The decrease in microglial SMEK1 causes it to secrete more pro-inflammatory factors, polarizing microglial cells in the M1 direction. The SMEK1 overexpression has proved to have the effect of improving the nerve injury after the cerebral arterial thrombosis of mice. In addition, the research of the invention finds that SMEK1 can regulate the polarization direction of microglial cells through mitochondrial metabolism reprogramming, and can reduce inflammatory response after ischemic cerebral apoplexy by promoting the expression of the SMEK 1. In a word, the invention provides a new mechanism research for the occurrence and development of ischemic cerebral apoplexy and provides a promising treatment strategy for ischemic cerebral apoplexy patients, thus having good practical application value.
Description
Technical Field
The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to application of SMEK1 in diagnosis and treatment of ischemic cerebral apoplexy.
Background
The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Cerebral Stroke (Stroke) has become the world's second leading cause of death next to ischemic heart disease, the chinese leading cause of death. Wherein ischemic cerebral apoplexy accounts for 80%. Ischemic stroke (Ischemic stroke) refers to the general term for brain tissue necrosis caused by cerebral arterial stenosis or occlusion and cerebral blood supply insufficiency, and is characterized by high morbidity, high disability rate and high mortality rate.
However, there are still limitations in the current therapy of AIS due to the narrow window of thrombolytic therapy after acute ischemic stroke (Acute ischemic stroke, AIS) and the adverse effects of reperfusion injury. Neuroinflammation and immune response occurring several minutes to hours after AIS are closely related to brain injury after AIS, and many preclinical and clinical studies on stroke indicate that immunomodulation of the central nervous system may be a viable alternative therapeutic strategy for AIS.
Disclosure of Invention
In view of the above prior art, an object of the present invention is to provide an application of SMEK1 in diagnosis and treatment of ischemic stroke. The invention discovers that the protein phosphatase 4 regulatory subunit 3a (SMEK 1) shows fluctuation change after ischemic cerebral apoplexy and has a descending trend for a quite long time. The decrease in microglial SMEK1 causes it to secrete more pro-inflammatory factors, polarizing microglial cells in the M1 direction. SMEK1 overexpression has been shown to have an effect of ameliorating nerve damage following ischemic stroke. Furthermore, the present study found that SMEK1 can modulate the polarization direction of microglial cells by mitochondrial metabolism reprogramming. Inflammatory responses after ischemic stroke can be reduced by promoting the expression of SMEK 1. Based on the above results, the present invention has been completed.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the invention, there is provided the use of a reagent for detecting a SMEK1 encoding gene and/or its expression product in the manufacture of a product for screening, (co) diagnosing, detecting, monitoring or predicting the progression of ischemic stroke.
In a second aspect of the invention, there is provided a system for screening, (co) diagnosing, detecting, monitoring or predicting the progression of ischemic stroke, the system comprising:
an acquisition module configured to: obtaining the expression level of a SMEK1 encoding gene and/or its expression product of a subject;
an evaluation module configured to: and judging the disease condition of the subject according to the expression level of the SMEK1 coding gene and/or the expression product thereof obtained by the obtaining module.
In a third aspect of the invention, there is provided a computer readable storage medium having stored thereon a program which when executed by a processor performs the functions of the system according to the second aspect of the invention.
In a fourth aspect of the invention, there is provided an electronic device comprising a memory, a processor and a program stored on the memory and executable on the processor, the processor implementing the functions of the system according to the second aspect of the invention when executing the program.
In a fifth aspect of the invention, there is provided the use of SMEK1 as a target in the manufacture and/or screening of a medicament for ischemic stroke.
The effect of the drug candidate on SMEK1 before and after use can be used to determine whether the drug candidate can be used to prevent or treat ischemic stroke.
In a sixth aspect of the invention there is provided the use of a substance which promotes the increase in the activity of the SMEK1 gene and its expression products in at least one of the following 1) -4):
1) Preparing a product for improving ischemic stroke mediated nerve injury;
2) Preparing a product for reducing inflammatory reactions mediated by ischemic stroke;
3) Inhibiting polarization of microglial cells to a pro-inflammatory direction (M1 type) after ischemic stroke or preparing a product for inhibiting polarization of microglial cells to a pro-inflammatory direction after ischemic stroke;
4) And preparing a product for preventing and/or treating cerebral arterial thrombosis.
Wherein the ischemic stroke may be an acute ischemic stroke.
The product can be a medicine or an experimental reagent, and the experimental reagent can be used for basic research, such as construction of cells or animal models related to ischemic cerebral apoplexy, so as to research the occurrence and development mechanisms of related diseases such as ischemic cerebral apoplexy and the like.
In a seventh aspect of the invention, there is provided a method of ischemic stroke treatment, the method comprising: administering to the subject a substance that promotes expression and/or increases activity of a SMEK1 encoding gene and its expression product.
The beneficial technical effects of one or more of the technical schemes are as follows:
the technical proposal reports that SMEK1 presents fluctuation change after ischemic cerebral apoplexy for the first time and presents descending trend in a quite long time. The decrease in microglial SMEK1 causes it to secrete more pro-inflammatory factors such as IL-1β, iNOS and TNF- α, which polarizes microglial cells in the M1 direction. The SMEK1 overexpression has proved to have the effect of improving the nerve injury after the cerebral arterial thrombosis of mice. Furthermore, the present study found that SMEK1 can modulate the polarization direction of microglial cells by mitochondrial metabolism reprogramming. The inflammatory reaction after the ischemic cerebral apoplexy can be reduced by promoting the expression of the SMEK1, and the functions of protection and treatment are achieved.
The technical scheme provides a new mechanism research for the occurrence and the development of the ischemic cerebral apoplexy and a promising treatment strategy for ischemic cerebral apoplexy patients, so that the method has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows the fluctuation of Smek1 expression in mouse brain tissue with time after MCAO/OGD according to an embodiment of the present invention. Wherein A and B show the processing time of MCAO/OGD. C and D show verification of MCAO modeling success. E shows the material-drawing part of the subsequent experiment. F and G show mRNA expression and Western blotting of brain tissue SMEK1 after MCAO. H and I show immunofluorescent staining of total SMEK1 and intra-microglial SMEK1 in brain tissue following MCAO.
FIG. 2 shows the fluctuation of Smek1 expression in mouse microglia of origin with time after MCAO/OGD according to the examples of the present invention. Wherein a and B show the results of flow cytometry staining of SMEK1 expression in microglia following MCAO. C, D, E, F show mRNA expression and Western blotting of SMEK1 after OGD/R of BV2 cells. G shows immunofluorescent staining of SMEK1 after OGD/R of BV2 cells. H and I show validation of mRNA levels and protein levels of BV2 cell knockdown/overexpression SMEK 1. J shows Western blotting of SMEK1 and IL-1β after knockdown of BV2 cells OGD of SMEK 1.
FIG. 3 is a graph showing that the decrease in microglial Smek1 after MCAO/OGD is associated with inflammation in accordance with an embodiment of the present invention. Wherein A and I show mRNA expression levels of IL-1β and IL-10 in BV2 cells knocked down/overexpressed SMEK1 in the absence of OGD/R stimulation. B, C, D, E, F, G, H show mRNA expression levels of CD86, IL-1β, iNOS, TNF- α, CD206, IL-10 and TGF- β, respectively, in BV2 cells knocked down SMEK1 after OGD/R stimulation. J, K, L, M, N, O, P show mRNA expression levels of CD86, IL-1β, iNOS, TNF- α, CD206, IL-10 and TGF- β, respectively, in BV2 cells overexpressing SMEK1 following OGD/R stimulation.
FIG. 4 is a graph showing the involvement of Smek1 in microglial cells in protecting nerve damage caused by ischemia in accordance with an embodiment of the present invention. Wherein a and B show verification of mRNA levels and protein levels of whole-body over-expressed SMEK1 transgenic mice. C shows the scoring of mNSS on the first, second and third days of MCAO before MCAO in the over-expressed transgenic mice. D shows cerebral cortical blood flow status of the MCAO on the first, second and third days before MCAO in the over-expressed transgenic mice. E, F, G, H, I, J, K show the results of flow cytometry staining of microglial cells expressing inflammatory factors after overexpression of transgenic mice MCAO.
FIG. 5 shows the results of transcriptome sequencing of BV2 cells after knockdown of SMEK1 in accordance with an embodiment of the present invention. Wherein, A shows acute cluster analysis on samples, which suggests that the correlation of the samples of biological repetition in the shNC group and the shSmek1 group is high. B and C show 594 up-regulated genes and 515 down-regulated genes in the transcriptome sequencing results. D shows enrichment of gp_bp into multiple cytokine-related biological processes suggesting that knock-down Smek1 affects BV2 cellular immune/inflammatory function. E shows the enrichment of gp_bp into multiple biological processes associated with lipid synthesis. G, H, I and F show GSEA functional assays, respectively, suggesting that SMEK1 knockdown is associated with fatty acid metabolism, cholesterol metabolism and pyruvate metabolism. J and K show mRNA levels and Western blots, respectively, of BV2 cells expressing PDK3 after knockdown/overexpression of SMEK 1.
FIG. 6 shows that deletion of Smek1 in microglial cells according to the examples of the invention results in reprogramming of mitochondrial metabolism. Wherein a and B show mRNA levels and western blots of HIF-1α expression following vev 2 cell OGD after knockdown/overexpression of SMEK 1. C, D, E show FAOBlue fatty acid oxidation assay results of BV2 cells after knockdown/over-expression of SMEK 1. F and G show extracellular and intracellular lactate levels, respectively, of BV2 cells after knockdown/overexpression of SMEK 1. H shows pyruvate dehydrogenase activity of BV2 cells after knockdown/overexpression of SMEK 1. I shows Western blots of pyruvate dehydrogenase and phosphorylated pyruvate dehydrogenase of BV2 cells after knockdown/overexpression of SMEK 1.
FIG. 7 shows that deletion of Smek1 in microglial cells according to the examples of the invention results in reprogramming of mitochondrial metabolism. Wherein a shows lactate dehydrogenase activity of BV2 cells after knockdown/overexpression of SMEK 1. B and C show OCR levels of BV2 cells after knockdown/overexpression of SMEK 1. D, E, F, G, H show the change in basal respiration, maximum respiration, proton leak, ATP production and reserve respiration amounts, respectively, of BV2 cells after knockdown of SMEK 1. I, J, K show the change in basal respiration, proton leak and ATP production, respectively, of BV2 cells after overexpression of SMEK 1. L and M show ECAR levels in BV2 cells after knockdown/overexpression of SMEK 1. N, O, P, Q show the change in basal glycolysis, basal proton efflux, plasmid efflux of glycolysis and acidification upon addition of 2-DG in BV2 cells after knockdown of SMEK 1.
FIG. 8 shows that deletion of Smek1 in microglial cells according to the examples of the invention results in reprogramming of mitochondrial metabolism. Wherein A and B show the change in basal glycolysis of BV2 cells after overexpression of SMEK1 and acidification after addition of 2-DG.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
As previously mentioned, immunomodulation of the central nervous system may be a viable alternative therapeutic strategy for AIS. Microglia are resident immune cells in the brain, which are activated and migrate to infarct core and penumbra areas the first time after an ischemic stroke occurs. Activation of microglia is generally thought to have two pathways, one being activation into pro-inflammatory microglia, the M1 type, by the "classical pathway; the other is activation into anti-inflammatory microglia, i.e. form M2, by an "alternative pathway". The surface mark of the M1 type microglial cell is CD86, and the pro-inflammatory factors such as IL-1 beta, TNF-alpha, IFN-gamma, IL-6, iNOS, MMP-9, MMP-3 and the like are secreted; the surface marker of M2 microglial cells is CD206, and secrete IL-10, TGF-beta, insulin-like growth factors, vascular Endothelial Growth Factor (VEGF) and other anti-inflammatory factors. After ischemic stroke, the change of micro-environment in brain activates microglial cells to two extreme, and the proportion imbalance of M1/M2 microglial cells plays a role in subsequent inflammatory reaction. In recent years, metabolic reprogramming has been increasingly emphasized by researchers. Metabolic reprogramming is a key driver of microglial immune responses, with microglial cells in a pro-inflammatory state preferentially using glycolysis to produce energy, while cells in an anti-inflammatory state are primarily powered by oxidative phosphorylation and fatty acid oxidation. Therefore, we want to influence the polarization of microglial cells towards M2 type by metabolic reprogramming, and thus play a neuroprotective role in inflammatory reactions after ischemic stroke.
Protein phosphatase 4 (PP 4) is a highly conserved serine/threonine phosphatase, a protein complex consisting of catalytic and regulatory subunits. MEK1 inhibitors (SMEK 1) are regulatory subunits of PP4 enzymes that regulate the activity of PP4 catalytic subunits, leading to dephosphorylation of their target substrates by unknown mechanisms. PP4 is involved in many cellular processes in organisms and regulates a variety of cellular signaling pathways including nuclear factor κb (NF- κb), c-Jun N-terminal kinase, apoptosis signaling, insulin receptor substrate protein 4, and rapamycin targets. Recent research results indicate that PP4 is an important regulator of T cell proliferation and immune response, activating protein kinase (AMPK) by dephosphorylation adenosine. In addition, CD4-Cre PP4fl/fl mice spontaneously develop rectal prolapse and colitis (similar to Crohn's disease) due to impaired regulatory T cell function. As a subunit of PP4, it is important to explore the role of SMEK1 in inflammatory responses after ischemic stroke, both theoretically and clinically.
In view of this, in a typical embodiment of the invention, there is provided the use of an agent for detecting a SMEK1 encoding gene and/or its expression product for the preparation of a product for screening, (co) diagnosing, detecting, monitoring or predicting the progression of ischemic stroke.
The research of the invention finds that SMEK1 shows fluctuation change after ischemic cerebral apoplexy and shows descending trend in a quite long time. Thus, the SMEK1 is closely related to the occurrence and the development of the cerebral arterial thrombosis.
In the present invention, the expression product of the SMEK1 encoding gene may obviously be a SMEK1 protein, which is a regulatory subunit of protein phosphatase 4 (PP 4).
The ischemic stroke progression includes but is not limited to ischemic stroke patients with acroparalysis, sensory deficit, loss of reflex, and cognitive dysfunction.
In the present invention, the ischemic stroke may be specifically an acute ischemic stroke.
The reagents for detecting the SMEK 1-encoding gene and/or its expression product include, but are not limited to, reagents for detecting transcription of the SMEK 1-encoding gene based on a sequencing method, based on a quantitative PCR method, or based on a probe hybridization method; or reagents for detecting SMEK1 protein expression based on immunodetection methods (such as immunohistochemistry, ELISA, colloidal gold test strips, protein chips).
The product may be a kit, a detection device or a detection apparatus, etc., and is not particularly limited herein.
In yet another embodiment of the present invention, there is provided a system for screening, (aiding) diagnosis, detection, monitoring or prediction of the progression of ischemic stroke, the system comprising:
an acquisition module configured to: obtaining the expression level of a SMEK1 coding gene and/or an expression product thereof of a sample to be tested of a subject;
an evaluation module configured to: and judging the disease condition of the subject according to the expression level of the SMEK1 coding gene and/or the expression product thereof obtained by the obtaining module.
Wherein the ischemic stroke is an acute ischemic stroke.
The subject may be a human or non-human mammal (e.g., mouse, etc.), and the sample to be tested includes, but is not limited to, peripheral blood and brain-associated tissues and cells (e.g., microglia, neurons, astrocytes, oligodendrocytes, etc.).
In yet another embodiment of the present invention, a computer-readable storage medium is provided, on which a program is stored which, when executed by a processor, performs the functions of the system as described above.
In yet another embodiment of the present invention, an electronic device is provided that includes a memory, a processor, and a program stored on the memory and executable on the processor, the processor implementing the functions of the system as described above when the program is executed.
In yet another embodiment of the invention, there is provided the use of SMEK1 as a target in the preparation and/or screening of ischemic stroke drugs.
The effect of the drug candidate on SMEK1 before and after use can be used to determine whether the drug candidate can be used to prevent or treat ischemic stroke.
Specifically, the method for screening ischemic cerebral apoplexy drugs comprises the following steps:
1) Treating the system expressing and/or containing said SMEK1 with a candidate substance; setting a parallel control without candidate substance treatment;
2) After step 1) is completed, detecting the expression level of the SMEK1 in a system; if the SMEK1 expression level is significantly increased in a system treated with a candidate substance, compared to a parallel control, the candidate substance can be used as a candidate ischemic stroke drug.
In yet another embodiment of the present invention, the system may be a cell system, a solution system, a tissue system, an organ system, or an animal system.
In yet another embodiment of the present invention, the cells in the cell system may be microglia, neuronal cells, astrocytes and oligodendrocytes;
in yet another embodiment of the present invention, the tissue in the tissue system may be the cortex, substantia nigra, striatum, thalamus, and brainstem quilt;
in yet another embodiment of the present invention, the organ in the organ system may be a brain;
in yet another embodiment of the present invention, the animal in the animal system may be a mammal, such as
Rats, mice, guinea pigs, rabbits, monkeys, humans, etc.
In a further embodiment of the invention, there is provided the use of a substance which promotes the SMEK1 gene and its expression products and/or increases its activity in at least one of the following 1) -4):
1) Preparing a product for improving ischemic stroke mediated nerve injury;
2) Preparing a product for reducing inflammatory reactions mediated by ischemic stroke;
3) Inhibiting polarization of microglial cells to a pro-inflammatory direction (M1 type) after ischemic stroke or preparing a product for inhibiting polarization of microglial cells to a pro-inflammatory direction after ischemic stroke;
4) And preparing a product for preventing and/or treating cerebral arterial thrombosis.
Wherein the ischemic stroke may be an acute ischemic stroke.
According to the present invention, the concept of "prevention and/or treatment" means any measure suitable for the treatment of ischemic stroke related diseases, or for the prophylactic treatment of such a represented disease or of a represented symptom, or for the avoidance of recurrence of such a disease, e.g. after the end of a treatment period or for the treatment of symptoms of a disease that have already been developed, such as dyskinesia, cognitive dysfunction, etc., or for the pre-interventional prevention or inhibition or reduction of the occurrence of such a disease or symptom.
Such agents that promote SMEK 1-encoding genes and their expression products include, but are not limited to, agents that up-regulate SMEK1 expression and/or promote its activity using gene-specific mic-based techniques; promoters or lentiviruses that up-regulate SMEK1 expression; and also comprises a compound accelerant.
The product can be a medicine or an experimental reagent, and the experimental reagent can be used for basic research, such as construction of cells or animal models related to ischemic cerebral apoplexy, so as to research the occurrence and development mechanisms of related diseases such as ischemic cerebral apoplexy and the like.
According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
In yet another embodiment of the present invention, the pharmaceutically inactive ingredient comprises a pharmaceutically acceptable carrier, excipient and/or diluent. Such as pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and nonionic surfactants or lipids, pharmacologically harmless salts such as sodium chloride, flavoring agents, vitamins such as vitamin a or vitamin E, tocopherols or provitamins, antioxidants such as ascorbic acid, and stabilizers and/or preservatives for prolonging the use and shelf life of a pharmaceutical active ingredient or formulation, and other commonly used non-pharmaceutical active ingredients or adjuvants and additives well known in the art, and mixtures thereof.
The administration dosage form of the medicine can be solid oral preparation, liquid oral preparation or injection.
The drug administration type can be tablets, dispersible tablets, enteric coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powder, oral solution, small water injection for injection, freeze-dried powder injection for injection, large transfusion or small transfusion.
In yet another embodiment of the present invention, the subject to be administered can be human or non-human mammal, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.
In yet another embodiment of the present invention, there is provided a method of treating ischemic stroke, the method comprising: administering to the subject a substance that promotes expression and/or increases activity of a SMEK1 encoding gene and its expression product.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are noted, and are generally conducted under conventional conditions.
Examples
The experimental method comprises the following steps:
1.1 animals and transient middle cerebral artery occlusion surgery (tMCAO)
All mice used in this study were C57BL/6j male mice purchased from Vetong Liwa (Beijing, china). The feed was kept under specific pathogen-free conditions with a fixed 12 hour light/dark period and sufficient food and water was available. We have minimized the number of mice used and their suffering.
C57BL/6j male mice (21-25 g) at 8-10 weeks of age were anesthetized, supine placed under an animal body microscope, shaved with Mao Qibei skin with small animals, sterilized, and then left common carotid artery, external carotid artery, and internal carotid artery were isolated. A incision is made in the proximal section of the common carotid artery, a wire plug (Shenzhen Ruiwo, 21-25g gauge) is inserted into the internal carotid artery along the common carotid artery, and finally delivered to the middle cerebral artery opening and its blood flow blocked. After the completion of the surgery, the mice were placed on an animal heating blanket and their body temperature was maintained at 37.0±0.5 ℃. After 60 minutes of occlusion, the wire plug is removed and reperfusion is completed. The same procedure was performed on sham mice, but no plug was inserted. Mice were sacrificed 1 day, 3 days, and 7 days after surgery, respectively (fig. 1A).
1.2 construction of transgenic mice
CAG Pr-loxP-Stop-loxP-SMEK1 CDS-HA Tag-WPRE-pA is inserted into a ROSA26 site, a C57BL/6J mode mouse is prepared based on an EGE system developed by CRISPR/Cas9, and the ROSA26-Smek1f/f mouse is mated with a Ubc-Cre-ERT2 mouse to obtain the ROSA26-Smek1f/fUbc-Cre-ERT2 mouse.
1.3 cerebral cortical blood flow and 2,3, 5-triphenyltetrazolium chloride (TTC) staining
Cerebral cortex blood flow was measured on SHAM group and MCAO group mice using a zoom laser speckle blood flow imaging system (moorinstrumental company, moorfpi-2, uk). Briefly, after anesthetizing the mice, they were cut along their head midline to expose the skull, and then placed under the imager lens to observe their blood flow (fig. 1D).
Infarct volume was determined by 2,3, 5-triphenyltetrazolium chloride (TTC) staining (beijing solebao, G3005). The brain tissue was removed and fixed on a flat surface and quick-frozen in a-20 degree freezer for 20 minutes, and then cut into 4-6 pieces with a razor blade. TTC dye was added and placed in a 37 ° incubator for 40 minutes during which time inversion was performed and infarct volume was observed after staining (fig. 1C).
1.4 immunofluorescent staining
Brain tissue frozen sections were permeabilized with 0.3% Triton X-100 (Beijing Soy Bao, T8200) for 30 minutes at room temperature and then blocked with 10% donkey serum (Beijing Soy Bao, SL 050) for one hour at room temperature. The rabbit-derived SMEK1 (Sigma, HPA002568, USA) was incubated overnight in a 4℃refrigerator with mouse-derived IBA-1 (Wuhansaivil, GB 12105). After rinsing with PBS, frozen sections were stained with the corresponding secondary antibody in dark environment for 2 hours at room temperature. Finally, the nuclei were stained with DAPI (shanghai bi yun, P0131). Paraffin sections were subjected to dewaxing and antigen retrieval procedures prior to staining, the remaining steps being identical to those of frozen sections.
1.5 microglial cell isolation and flow cytometry
After heart infusion, the mice were sampled, brain tissue was directly ground and the suspension was filtered off, and the suspension and 100% percoll (beijing solebao, P8370) were formulated as 30% percoll. The 30% percoll containing suspension was slowly added to the 70% percoll liquid surface and the cells were separated by density gradient centrifugation (700 g,25 min, minimum deceleration) and the middle buffy coat was aspirated into a centrifuge tube for use. The single cell suspension was stained with Zombie yellow dye-BV605 (Biolegend, b 337269) dye at room temperature for 10 minutes, and then stained with CD45-APC-CY7 flow antibody (Biolegend, 103116) and CD11b-BV421 (Biolegend, 101236) dye at 4℃for 30 minutes. Foxp3 fixative (U.S. Sieimer, 00-5523-00) was used to fix for 45 minutes at room temperature and Foxp3 wash (U.S. Sieimer, 00-5523-00) was rinsed twice. TNF-alpha-PE (Biolegend, 504104), iNOS-PECy7 (eBioscience, 25-5920-82), IL-10-APC (Biolegend, 505010) and rabbit SMEK1 (Sigma, HPA 002568) were added and incubated for 45 min at room temperature and stained with goat anti-rabbit 488-FITC (Abcam, ab 150073) for 45 min at room temperature. Finally, 300ul of 0.5% multimerization was used
Formaldehyde (Wuhansai Weir, G1101) resuspended cells and were detected on-machine. All dyes are used in the corresponding instructions.
1.6 transcriptome sequencing analysis
BV2 microglia knocked down from SMEK1 were collected and washed once with PBS, then lysed in TRIzol at room temperature for 5 min, and frozen at-80 ℃. Samples were sent to beijing norelvan for library preparation and sequencing. The original data in fastq format is first processed by the internal Perl script as a low quality read. After localization to the reference genome by Hisat2 (v 2.0.5), FPKM was calculated based on the number of reads and length of each gene. In addition, differential expression analysis was performed on both groups using the DESeq 2R software package (version 1.20.0). In order to control the error occurrence rate, p values are adjusted by adopting methods of Benjamini and Hochberg, and the panj is not more than 0.05 and the log2 (fold change) is not less than 1, which are considered to have statistical significance. Gene Ontology (GO) enrichment analysis was performed on differentially expressed genes using the clusterProfiler R software package (3.8.1).
1.7 cell culture and lentiviral transfection
Mouse BV2 microglia (Bodhisattva, CX 0103) were cultured in Dulbecco's modified Eagle's medium (DMEM, gibco, U.S. Pat. No. 3, 11995500 BT) supplemented with 10% fetal bovine serum (Gibco, U.S. Pat. No. 3, 3161001C) and 1% penicillin-streptomycin (Gibco, U.S. Pat. No. 3, 15070063). Cells were incubated at 37℃in a 5% carbon dioxide incubator with medium changed every 2 days.
For knockout or overexpression of SMEK1 in BV2 cells, target gene fragments were amplified with the following primers: 49830FW-65237 GCGAATTCGAAGTATATAACCTCGAGGC; 49830RW-65238 CGATCGCAGGACTCCTTGGATCC. This fragment was then cloned into the lentiviral vectors PGMLV-hU6-MCS-CMV-Puro-WPRE and lenti-CMV-MCS-PGK-Puro (Shanghai, genodiech). Following successful construction, lentiviruses containing the corresponding target fragment or negative control are transfected into BV2 cells. The medium was aspirated after 48 hours of transfection and incubated for another 48 hours, and BV2 cells knocked out or overexpressed SMEK1 were selected with puromycin (2.5. Mu.g/ml; shanghai MCE, HY-B1743A/CS-6857). Verification was performed using RT-qPCR and Western Blots (FIG. 2H, I).
1.8 oxygen sugar deprivation (OGD) experiments
To simulate ischemic stroke in vitro, cells were subjected to OGD/R treatment. Briefly, glucose-free DMEM (Gibco, 11966025) was used instead of normal medium and incubated in a hypoxic incubator (94% nitrogen and 5% carbon dioxide) at 37 ℃ for 3 hours to simulate OGD damage. The medium was then changed to DMEM medium containing normal glucose and the cells transferred to a normal incubator for reoxygenation for 1h, 6h, 12h, 21h and 24h, respectively (fig. 1B).
1.9SeaHorse experiment
BV2 cells knocked down/overexpressed SMEK1 were cultured at 2X 105 cells/ml for 2 days and then re-seeded at 4X 104 cells/well in SeaHorse 24-well microplates. The metabolic status of the cells was measured using the Seahorse XF cell Mito pressure test kit (U.S. Agilent Technologies, 103015-100), the Seahorse XF glycolysis rate assay kit (U.S. Agilent Technologies, 103344-100) and the Seahorse XF24 extracellular flux analyzer. To determine oxygen consumption, we sequentially added mitochondrial complex inhibitors (oligosomycin [ 1. Mu.M ], FCCP [ 1. Mu.M ], rotenone/antimycin A [ 0.5. Mu.M ]) to cell culture microplates. To measure the glycolytic rate, we sequentially added rotenone/antimycin A [ 0.5. Mu.M ] and 2-deoxy-D-glucose [50mM ] to cell culture microplates. Key parameters of cell metabolism were measured using a Seahorse XF24 analyzer. At least three replicates were performed for each sample. The results were analyzed using Wave software (U.S. Agilent Technologies).
1.10 lactic acid level determination
The kit for measuring the lactic acid level is an L-lactic acid (L-LA) content detection kit (Beijing Soy Bao, BC 2235). The treatment and experimental procedures for the cells or supernatant were performed according to the kit instructions. Absorbance was measured at 570nm using a microplate reader (BioTek, epoch, usa).
1.11 detection of pyruvate dehydrogenase Activity and lactate dehydrogenase Activity
The kits for measuring the activity of pyruvate dehydrogenase and lactate dehydrogenase are a Pyruvate Dehydrogenase (PDH) activity detection kit (BC 0385, soy baby, beijing) and a Lactate Dehydrogenase (LDH) activity detection kit (BC 0685, soy baby, beijing), respectively. The treatment and experimental procedures for the cells were performed according to the kit instructions. Absorbance was measured at 605nm and 450nm, respectively, using a microplate reader (BioTek, epoch, usa).
1.12FAOBlue fatty acid Oxidation assay
We used FAOBlue fatty acid oxidation assay reagent (Funakoshi, japan, FDV-0033) to stain BV2 cells to detect their level of fatty acid oxidation. Briefly, the reagent was added to the medium and incubated at 37℃for 2 hours. The average fluorescence intensity was recorded on a flow cytometer (Beckmann, U.S.A.A.00-1-1102) with confocal microscopy (Nikon, LU-N4) at 405nm wavelength or with PB450 channels.
1.13 real-time fluorescent quantitative PCR
For extraction of total RNA from BV2 cell lines we used the RNA flash extraction kit (beijing root, DP 451). For extraction of total RNA from brain tissue, we used TRIzol reagent (Sieimer, america 15596026). The extraction steps are respectively carried out according to corresponding specifications. The resultant RNA was then reverse transcribed into cDNA using a reverse transcription kit (Japanese Takara, RR 047A) according to the instructions. qPCR reactions were performed on a real-time PCR detection system (Bio-Rad, CFX96 Optics Module, U.S.A.) using FastStart Universal SYBR Green Master Mix (Nanjinopran, Q321-02). The primers used for amplification were as follows:
1.14 Western blotting
Brain tissues and cultured cells were lysed with RIPA lysate (shanghai bi-cloudy, P0013B) supplemented with PMSF (beijing solibao, P0100) and phosphatase inhibitor (shanghai bi-cloudy, P1081). Proteins were separated by electrophoresis on a 10% SDS-PAGE gel (Shanghai elegance enzyme, PG 112) and then transferred to PVDF membrane (Merck, germany, no. ISEQ 00010). After blocking with a rapid blocking solution (Shanghai Genefist, GF 1815), the PVDF membrane was incubated with the primary antibody overnight at 4 ℃. Primary antibodies used included SMEK1 (1:2000, U.S. Sigma, HPA 002568), β -actin (1:20000, wohan three eagles, 66009-1-Ig), β -tubulin (1:1000, wohan three eagles, 10068-1-AP), PDH (1:6000, wohan three eagles, 18068-1-AP), P-PDH (1:2000, abcam, ab 177461), HIF-1α (1:1000, abcam, ab 179483), PDK3 (1:500, wohan three eagles, 12215-1-AP), IL-1β (1:1000, U.S. CST, 31202), CD206 (1:1000, U.S. CST, 24595). After washing, PVDF membranes were incubated with a secondary antibody of appropriate primary antibody source species for 2 hours at room temperature. Finally, immunoblots were tested using a hypersensitive ECL chemiluminescent detection kit (wuhan Sanying, PK 10003). Protein bands were visualized using a chemiluminescent fully automated imaging system (Bio-Rad, chemi Doc, U.S.A.).
1.15 mouse behavioural detection
Mice were comprehensively tested for motor, sensory, reflex and balance functions using a modified nervous system severity score (mNSS) test to assess overall neurological deficit. Scores range from 0 to 18, with 0 representing normal and 18 representing the most severe defect. Functional defects were assessed 1 day, 2 days and 3 days post-surgery, respectively. Experimental results:
we first examined SMEK1 expression in total mRNA and protein in mouse brain tissue following MCAO. The results showed that Smek1 peaked on day 1 after MCAO and decreased expression on days 3-7, with minimal decrease in Smek1 expression on day 3 after MCAO (fig. 1f, g). Immunofluorescent staining demonstrated reduced expression of Smek1 in brain tissue of mice on the third day after MCAO (fig. 1H). Further, we demonstrated reduced expression of SMEK1 in microglia cells on day 3 after MCAO using immunofluorescent staining and flow cytometry (fig. 1I, fig. 2a, b). Likewise, the expression of SMEK1 in mRNA and protein of murine BV2 microglia was examined in vitro after OGD. The results show a trend of increasing and decreasing Smek1 expression in BV2 cells following OGD with prolonged reoxygenation time, with minimal decrease in Smek1 expression for OGD3h/R21h (fig. 2c, d, e, f). Immunofluorescent staining demonstrated reduced Smek1 expression by BV2 cells at OGD3h/R21h (fig. 2G). Our findings demonstrate a trend of increasing and then decreasing expression of Smek1 in microglia following MCAO over time and a trend of decreasing over a considerable period of time. We speculate that Smek1 within microglia may be involved in immune regulation after ischemic stroke.
To determine whether Smek1 in microglial cells is involved in immune regulation after ischemic stroke, we constructed BV2 cell stably transformed cell lines knocked down/overexpressed Smek1 using lentiviral transfection techniques. Interestingly, the expression of the pro-inflammatory factor IL-1β was increased in the case of BV2 cells knockdown with Smek1 group without hypoxia stimulation (fig. 3A). The expression of the anti-inflammatory factor IL-10 was increased in the BV2 cells of the Smek1 group without hypoxia stimulation (FIG. 3I). Compared to the negative control group (shNC), the Smek1 knockdown group (shSMEK 1) BV2 cells showed increased expression of CD86, IL-1β, iNOS and TNF- α after OGD3h/R21h (FIG. 2J, FIG. 3B, C, D, E), and decreased expression of CD206, IL-10 and TGF- β (FIG. 3F, G, H). The Smek1 over-expression group (oe-SMEK 1) BV2 cells showed increased expression of CD206, IL-10 and TGF- β (FIG. 3N, O, P) and decreased expression of CD86, IL-1β, iNOS and TNF- α (FIG. 3J, K, L, M) after OGD3h/R21h compared to the negative control group (oeNC). The results show that Smek1 in microglial cells is involved in immune regulation after ischemic cerebral apoplexy and plays a role in neuroprotection.
To study the effect of microglial SMEK1 overexpression on post-ischemic stroke inflammation, we performed intraperitoneal injection of tamoxifen (100 mg/kg, sigma, T5648 in the united states) on 8 SMEK1 overexpressing mice to induce SMEK1 overexpression, intraperitoneal injection of corn oil on the other 8 SMEK1 overexpressing mice as a control, and then performed MCAO surgery on them. mNSS scoring and cerebral cortex blood flow imaging were performed 1 day, 2 days and 3 days after MCAO, respectively. Mice were sacrificed the third day after MCAO and mononuclear cells from the infarct zone were extracted for flow staining while the expression of SMEK1 was verified by extracting RNA from the side brain tissue and protein. We validated the overexpression of SMEK1 by RT-qPCR and Western Blots (FIGS. 4A, B). The mNSS score was significantly reduced in SMEK1 over-expressed mice compared to control mice (fig. 4C), and the difference was most pronounced on the third day of MCAO, suggesting that SMEK1 has a potential neuroprotective effect on neurological recovery following ischemic stroke. The zoom laser speckle blood flow imaging system showed better brain blood flow recovery in SMEK1 over-expressed mice compared to control mice (fig. 4D). Flow cytometry showed that microglia overexpressing SMEK1 expressed IL-10 increased after MCAO (FIGS. 4J, K) and TNF- α and iNOS decreased (FIGS. 4F, G, H, I). The above results indicate that SMEK1 in microglia is involved in protecting nerve damage caused by ischemia.
To reveal the mechanism of action of Smek1, we performed transcriptome sequencing analysis on BV2 microglia knocked down Smek 1. In total 594 genes were up-regulated and 515 genes were down-regulated in the Smek1 group BV2 cells compared to the negative control group (fig. 5b, c), indicating that the knockdown of Smek1 alters the gene expression profile of microglia. Subsequently, we performed a Gene Ontology (GO) analysis on these differentially expressed genes. The results showed that Smek1 knockdown was closely related to fatty acid metabolism, cholesterol metabolism and pyruvate metabolism (fig. 5g, h, i). We have also found that the index pyruvate dehydrogenase kinase 3 (PDK 3) associated with carbohydrate metabolism is significantly increased after smok 1 knockdown.
We demonstrate by experiments that expression of PDK3 is significantly increased after smok 1 knock down and that PDK3 expression is decreased after smok 1 overexpression (fig. 5j, k). To further elucidate why PDK3 expression increases after SMEK1 knockdown, we consult the literature to find that hypoxia induces PDK3 expression by upregulating hypoxia inducible factor-1α (HIF-1α), and that PDK3 expression is positively correlated with HIF-1α expression. Next, we examined HIF-1α expression after hypoxia. The results show increased HIF-1α expression following Smek1 knockdown. Expression of HIF-1α was reduced after overexpression of Smek1 (fig. 6a, b). This result is consistent with the change in PDK 3.
To demonstrate that the mitochondrial metabolism was altered after the smok 1 knock down in microglial cells, we examined the Fatty Acid Oxidation (FAO) level and the intracellular and extracellular Lactic Acid (LA) content, respectively. The results show that the FAO level is reduced after Smek1 knockdown, and the intracellular and extracellular lactic acid content is obviously increased. FAO levels increased after Smek1 overexpression and intracellular and extracellular lactate levels decreased (FIGS. 6C, D, E, F, G). Next, we examined PDK 3-regulated Pyruvate Dehydrogenase (PDH) involved in cellular metabolism. The results showed that PDH activity was decreased and phosphopyruvate dehydrogenase (P-PDH) expression was increased after the Smek1 knockdown. PDH activity increased and P-PDH expression decreased after Smek1 was overexpressed (FIG. 6H, I). Similarly, we also examined the activity of Lactate Dehydrogenase (LDH), which showed an increase in LDH activity after knockdown of Smek1 and a decrease in LDH activity after overexpression of Smek1 (fig. 7A). To further understand the changes in cell metabolism following knockdown/overexpression of microglial SMEK1, we used a Seahorse XF24 extracellular flux analyzer to detect Oxygen Consumption Rate (OCR) and extracellular acidification rate (ECAR), respectively, that reflect mitochondrial oxidative phosphorylation levels and glucose metabolism levels. The results showed that knocking down BV2 cell SMEK1 enhanced glycolysis levels (fig. 7L), enhanced basal glycolysis, basal proton efflux rates, glycolytic plasmid efflux rates and acidification upon addition of 2-DG (fig. 7n, o, p, q), attenuated mitochondrial oxidative phosphorylation levels (fig. 7B), attenuated basal respiration, maximum respiration, proton leakage, ATP production and reserve respiration (fig. 7d, e, f, g, h). Overexpression of BV2 cells SMEK1 increased mitochondrial oxidative phosphorylation (FIG. 7C), increased basal respiration, proton leakage and ATP production (FIG. 7I, J, K), decreased glycolysis (FIG. 7M), decreased basal glycolysis and acidification after 2-DG addition (FIG. 8A, B). This result indicates that upon knockdown of microglial cells Smek1, the metabolic state of the cells is altered, glycolytic levels are increased, and mitochondrial oxidative phosphorylation levels are decreased. Conversely, after overexpression of Smek1, microglial mitochondrial oxidative phosphorylation levels increased, while glycolysis levels decreased. These results indicate that deletion of Smek1 in microglia can lead to reprogramming of mitochondrial metabolism.
The invention proves that the deficiency of SMEK1 can lead microglial cells to polarize towards a pro-inflammatory direction after cerebral arterial thrombosis so as to exacerbate inflammation. SMEK1 modulates the polarization of microglial cells in the pro-inflammatory direction by reprogramming of mitochondrial metabolism. Our conclusion is that SMEK1 is critical for reducing inflammatory responses after ischemic stroke. Because of
In this way, increased SMEK1 expression may reduce inflammation in ischemic stroke and may be a viable treatment for ischemic stroke.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (4)
1. Use of a reagent for detecting SMEK1 encoding gene and/or its expression product for the preparation of a product for screening, diagnosing, detecting, monitoring or predicting the progression of ischemic stroke.
2. The use of claim 1, wherein the progression of ischemic stroke comprises palsy, sensory deficit, loss of reflex, and cognitive dysfunction in an ischemic stroke patient;
the ischemic stroke is specifically acute ischemic stroke.
3. The use according to claim 1, wherein the reagent for detecting the SMEK 1-encoding gene and/or its expression product comprises a reagent for detecting transcription of the SMEK 1-encoding gene based on a sequencing method, based on a quantitative PCR method or based on a probe hybridization method; or a reagent for detecting the expression of the SMEK1 protein based on an immunodetection method;
the product is a kit, a detection device or detection equipment.
4. Use of a substance that promotes SMEK1 gene expression and/or increases the activity of its expression product in at least one of the following 1) -4):
1) Preparing a product for improving ischemic stroke mediated nerve injury;
2) Preparing a product for reducing inflammatory reactions mediated by ischemic stroke;
3) Preparing a product for inhibiting the polarization of microglial cells to a pro-inflammatory direction after cerebral arterial thrombosis;
4) Preparing a product for preventing and/or treating ischemic cerebral apoplexy;
the substance promoting the expression of the SMEK1 gene and improving the activity of an expression product thereof comprises a promoter or a lentivirus for up-regulating the expression of the SMEK 1.
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