Application of YBX1 in preparation of medicine for treating or preventing myocardial infarction and vascular injury

Abstract

The invention discloses application of YBX1 in preparation of medicine for treating or preventing myocardial infarction and vascular injury, which is characterized in that the YBX1 is used as a biomarker for diagnosing myocardial infarction or vascular injury, the amino acid sequence of the YBX1 is as shown in SEQ ID NO: 1, injury caused by myocardial infarction can be aggravated by inhibiting expression of the YBX1, and heart injury caused by myocardial infarction can be inhibited by overexpression of the YBX1 in vivo; the invention further provides a diagnostic kit for myocardial infarction and vascular injury. The diagnostic kit comprises a reagent for detecting YBX1 expression. The invention further provides application of the YBX1 to preparation of a medicine for preventing or treating myocardial infarction and vascular injury, and the medicine promotes in-vivo overexpression of the YBX1 and promotes human umbilical vein endothelial cell activity and angiogenesis. A new pathophysiology mechanism is provided for occurrence and development of myocardial infarction diseases, a new treatment target YBX1 is provided for myocardial infarction, and a new way is opened up for development of cardiovascular disease treatment methods.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of YBX1 gene or protein in the preparation of drugs for treating or preventing myocardial infarction and vascular damage, especially the application of YBX1 gene or protein in the preparation of drugs for treating or preventing vascular damage during the occurrence of myocardial infarction. Background Art

[0002] Myocardial infarction refers to a disease in which myocardial ischemia and necrosis occur due to coronary artery obstruction. It is one of the main causes of death from cardiovascular disease. At present, clinical treatments mainly include drug therapy, interventional therapy, and surgical treatment. However, these treatments have certain limitations. Drug therapy requires long-term use and has side effects. Interventional therapy and surgical treatment also have certain risks and complications. Therefore, finding more effective treatments has always been an important direction of myocardial infarction research. A deep understanding of the pathogenesis and pathophysiological process of myocardial infarction can provide a theoretical basis for the prevention and treatment of myocardial infarction and provide an important reference for the prevention and treatment of cardiovascular diseases.

[0003] Angiogenesis plays a very important role in myocardial infarction. After myocardial infarction, myocardial tissue will experience a period of ischemia due to insufficient blood supply. Angiogenesis, also known as vasculogenesis, is a natural repair process that restores blood supply to damaged tissues through the formation of new blood vessels. The main effects of angiogenesis include: (1) restoring blood flow. New blood vessels can bypass the damaged coronary artery area and provide the myocardium with necessary oxygen and nutrients, thereby helping to alleviate the ischemic state; (2) reducing the infarct area, protecting more myocardial tissue from damage, providing nutrients and oxygen to myocardial cells, and promoting the repair and regeneration of myocardial tissue. Although angiogenesis has a positive effect after myocardial infarction, the process is complex and is regulated by multiple factors. It is of great significance to explore how to promote or regulate angiogenesis through drugs or other interventions to improve the prognosis of patients with myocardial infarction.

[0004] Y-box-binding protein 1 (YBX1) is a member of the cold shock domain protein family and has multiple biological functions, including affecting cell proliferation, differentiation, and responding to external stress. As a DNA and RNA binding protein, YBX1 is involved in multiple cellular processes such as transcription and translation regulation and cell proliferation. In cancer development, YBX1 can promote tumor growth and metastasis through different molecular mechanisms. In heart disease, YBX1 can interact with microRNA, circRNA, and lncRNA to regulate cell growth and protein synthesis, and is required for the growth of pathological cardiomyocytes. Although studies have shown a link between YBX1 and cardiovascular disease, its specific role and mechanism in myocardial infarction, especially in angiogenesis, remain unclear. Summary of the invention

[0005] In order to solve the above problems and overcome the deficiencies of existing myocardial infarction-related research and treatment technologies, the purpose of the present invention is to provide the use of YBX1 gene or YBX1 protein in the preparation of drugs for preventing or treating myocardial infarction and vascular injury. First, it is proved that YBX1 expression is significantly upregulated in experimental myocardial infarction mice and hypoxic human umbilical vein endothelial cells, overexpression of YBX1 increases the vitality and proliferation ability of human umbilical vein endothelial cells and promotes angiogenesis, and knockdown of YBX1 inhibits the vitality, proliferation ability and angiogenesis of human umbilical vein endothelial cells. Secondly, by using adeno-associated virus 9 (AAV9) to encapsulate YBX1 and overexpress YBX1 in myocardial infarction-induced mice after MI surgery, the damage caused by myocardial infarction can be effectively alleviated, the infarct area of ​​mouse heart tissue can be reduced, and angiogenesis can be promoted, thereby confirming that YBX1 has a significant and stable protective effect on ischemic heart. The present invention confirms that YBX1 can be used as a new drug or drug target, which can be used to prevent and treat myocardial infarction and vascular injury.

[0006] The above-mentioned object of the present invention can be achieved by the following technical solutions:

[0007] According to a first aspect of the present invention, YBX1 is used as a biomarker for diagnosing myocardial infarction and vascular injury. The amino acid sequence of YBX1 is shown in SEQ ID NO: 1, specifically: MSSEAETQQPPAAPAAALSAADTKPGSTGSGAGSGGPGGLTSAAPAGGDKKVIATKVLGTVKWFNVRNGYGFINRNDTKEDVFVHQTAIKKNNPRKYLRSVGDGETVEFDVVEGEKGAEAANVTGPGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNYQNSESGEKNEGSESAPEGQAQQRRPYRRRRFPPYYMRRPYARRPQYSNPPVQGEVMEGADNQGAGEQGRPVRQNMYRGYRPRFRRGPPRQRQPREDGNEEDKENQGDETQGQQPPQRRYRRNFNYRRRRPENPKPQDGKETKAADPPAENSSAPEAEQGGAE.

[0008] Preferably, inhibiting the expression of YBX1 can aggravate the damage caused by myocardial infarction; overexpressing YBX1 in vivo can inhibit the heart damage caused by myocardial infarction, thereby protecting the heart.

[0009] In a second aspect, the present invention provides a diagnostic kit for myocardial infarction and vascular injury, comprising a reagent for detecting the expression of YBX1, wherein the amino acid sequence of YBX1 is shown in SEQ ID NO:1.

[0010] The third aspect of the present invention provides use of YBX1 in the preparation of a drug for preventing or treating myocardial infarction and vascular injury, wherein the amino acid sequence of YBX1 is shown in SEQ ID NO:1.

[0011] Preferably, the drug can achieve in vivo overexpression of YBX1, promoting the activity and angiogenesis of human umbilical vein endothelial cells.

[0012] In a fourth aspect of the present invention, a reagent for detecting the expression of YBX1 is used in the preparation of a drug for preventing or treating myocardial infarction and vascular injury, wherein the amino acid sequence of YBX1 is shown in SEQ ID NO:1.

[0013] In a fifth aspect, the present invention provides use of a YBX1 activator in the preparation of a drug for preventing or treating myocardial infarction and vascular injury, wherein the amino acid sequence of the YBX1 is shown in SEQ ID NO:1.

[0014] In a sixth aspect, the present invention provides a YBX1-specific siRNA molecule, whose targeting sequence is shown in SEQ ID NO:2.

[0015] In a seventh aspect, the present invention provides a YBX1 overexpression plasmid vector, which carries the overexpressed YBX1 gene, and the vector used is an adeno-associated virus type 9 vector (AAV9).

[0016] The eighth aspect of the present invention provides the use of the YBX1-specific siRNA molecule or the YBX1 overexpression plasmid vector in the preparation of a drug for preventing or treating myocardial infarction and vascular injury.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention first discovered the important role of YBX1 gene or protein in the treatment of myocardial infarction and vascular injury, and revealed the mechanism by which YBX1 improves cardiac function by promoting the viability and angiogenesis of human umbilical vein endothelial cells. Experiments have shown that YBX1 expression is significantly upregulated in experimental myocardial infarction mice and hypoxic human umbilical vein endothelial cells, overexpression of YBX1 increases the viability and proliferation of human umbilical vein endothelial cells and promotes angiogenesis, while knocking down YBX1 inhibits the viability and proliferation of human umbilical vein endothelial cells and inhibits angiogenesis.

[0019] 2. The present invention uses adeno-associated virus 9 carrying YBX1 plasmid to infect mice at the in vivo level to overexpress the YBX1 gene and construct a myocardial infarction surgery model. The results show that overexpression of YBX1 significantly reduces the left ventricular infarction area of ​​mice in the MI model group, significantly increases the left ventricular ejection fraction and shortening rate, improves the cardiac function of mice, and can inhibit the heart damage caused by myocardial infarction surgery, proving the potential medical value of YBX1 as a new drug target and providing an innovative strategy for the treatment of myocardial infarction.

[0020] 3. The present invention provides a new pathophysiological mechanism for the occurrence and development of myocardial infarction, confirming that YBX1 has a significant and stable protective effect on ischemic heart and promotes angiogenesis. The present invention not only provides a new therapeutic target YBX1 for myocardial infarction, but also demonstrates the potential application of angiogenesis induced by YBX1 in actual medical treatment, opening up a way to develop new treatment methods for cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of the expression changes of YBX1 in the heart of myocardial infarction (MI) mice in the examples are shown; wherein: Figure 1 A is the immunofluorescence staining result of the left ventricular infarct edge area tissue of mice 28 days after the successful establishment of the MI model; Figure 1 B is the expression of YBX1 in vascular endothelial cells, n=5, P=0.0054.

[0022] Figure 2The results of the effects of YBX1 on human umbilical vein endothelial cells (HUVEC) under hypoxia in the examples are shown; wherein: Figure 2 A is the growth curve of HUVEC cells measured by CCK-8 method under the condition of overexpression of YBX1; Figure 2 B shows the effect of knocking down YBX1 on HUVEC cell growth; Figure 2 C is the result of cell proliferation imaging analysis (EDU staining), red represents proliferating cells, blue represents cell nuclei; the right side is the statistical graph of EDU results, n=5, P<0.0054 vs YBX1-NC, P=0.0027 vs si-YBX1-NC; Figure 2 D is the result of angiogenesis in HUVECs, n=5, P=0.0004 vs YBX1-NC, P=0.0137 vs si-YBX1-NC, the data are expressed as mean±SEM.

[0023] Figure 3 The example shows the effect of overexpression of YBX1 on myocardial infarction and damaged blood vessels in mice after in vivo injection of AAV9 virus carrying YBX1 in the sham operation group or after MI surgery; wherein: Figure 3 A is the result of detecting cardiac function of mice in each group by M-mode ultrasound after MI or Sham surgery; Figure 3 B is a statistical graph of left ventricular ejection fraction, shortening rate, left ventricular end-diastolic diameter and left ventricular end-systolic diameter; Figure 3 C is a Masson staining experiment to detect the content of collagen fibers, red is muscle fiber, and blue is collagen fiber; Figure 3 D is the statistical graph of Masson staining, n = 5, P = 0.0002; Figure 3 E is the immunofluorescence staining result of mouse heart tissue, green is CD31 positive tissue representing vascular endothelial cells, red is cTnT antibody representing cardiomyocytes, and blue is DAPI representing cell nuclei; Figure 3 F is the statistical graph of immunofluorescence staining.

[0024] Figure 4 The example shows the effect of knocking down YBX1 on myocardial infarction and damaged blood vessels in mice after in vivo injection of AAV9 virus carrying SH-YBX1 in sham or MI surgery; wherein: Figure 4 A is the result of detecting cardiac function of mice in each group by M-mode ultrasound after MI or Sham surgery; Figure 4 B is a statistical graph of left ventricular ejection fraction, shortening rate, left ventricular end-diastolic diameter and left ventricular end-systolic diameter; Figure 4 C is a Masson staining experiment to detect the content of collagen fibers, red is muscle fiber, and blue is collagen fiber; Figure 4D is the statistical graph of Masson staining, n = 5, P = 0.0119; Figure 4 E is the immunofluorescence staining result of mouse heart tissue, green is CD31 positive tissue representing vascular endothelial cells, red is cTnT antibody representing cardiomyocytes, and blue is DAPI representing cell nuclei; Figure 4 F is the statistical graph of immunofluorescence staining. DETAILED DESCRIPTION

[0025] In order to more fully understand and demonstrate the technical solutions, purposes and advantages of the present invention, the technical effects produced by the present invention are further described in detail and completely in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all. It should be pointed out that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention all belong to the protection scope of the present invention, and the present invention is specifically described in conjunction with the accompanying drawings and embodiments.

[0026] The following example provides an application of YBX1 as a biomarker for diagnosing myocardial infarction and vascular injury. The amino acid sequence of YBX1 is shown in SEQ ID NO: 1, namely: MSSEAETQQPPAAPAAALSAADTKPGSTGSGAGSGGPGGLTSAAPAGGDKKVIATKVLGTVKWFNVRNGYGFINRNDTKEDVFVHQTAIKKNNPRKYLRSVGDGETVEFDVVEGEKGAEAANVTGPGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNYQNSESGEKNEGSESAPEGQAQQRRPYRRRRFPPYYMRRPYARR PQYSNPPVQGEVMEGADNQGAGEQGRPVRQNMYRGYRPRFRRGPPRQRQPREDGNEEDKENQGDETQGQQPPQRRYRRNFNYRRRRPENPKPQDGKETKAADPPAENSSAPEAEQGGAE; among them, inhibiting the expression of YBX1 can aggravate the damage caused by myocardial infarction; overexpression of YBX1 in vivo can inhibit the heart damage caused by myocardial infarction, thereby protecting the heart.

[0027] The following example provides a diagnostic kit for myocardial infarction and vascular injury, comprising a reagent for detecting the expression of YBX1, wherein the amino acid sequence of YBX1 is shown in SEQ ID NO:1.

[0028] The following examples provide the use of YBX1 in the preparation of a drug for preventing or treating myocardial infarction and vascular injury. The amino acid sequence of YBX1 is shown in SEQ ID NO:1.

[0029] The following examples provide the use of a reagent for detecting the expression of YBX1 in the preparation of a drug for preventing or treating myocardial infarction and vascular injury. The amino acid sequence of YBX1 is shown in SEQ ID NO:1.

[0030] The following examples provide the use of a YBX1 activator in the preparation of a drug for preventing or treating myocardial infarction and vascular injury. The amino acid sequence of YBX1 is shown in SEQ ID NO:1.

[0031] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Unless otherwise specified in the following examples, all reagents and materials used were commercially available.

[0033] Example 1: YBX1 is highly expressed in the infarct margin area after myocardial infarction

[0034] In this example, myocardial infarction surgery was used to construct a mouse myocardial infarction experimental model. 6-8 weeks old healthy adult C57BL / 6 mice were selected, 1% Avertin at a dose of 0.1 mL / 10 g was anesthetized by intraperitoneal injection, the left anterior descending coronary artery (LAD) was ligated with 7-0 sutures, and the apex of the heart was observed to see if it was white, to preliminarily determine whether the model construction was successful. 28 days after myocardial infarction, mouse heart tissue was collected for pathological immunofluorescence staining, CD31 antibody was used to label vascular endothelial cells, and DAPI was used to label cell nuclei. The results showed that YBX1 expression was increased in the infarct edge area after myocardial infarction ( Figure 1 AB), indicating that YBX1 may be involved in the angiogenesis process of myocardial infarction.

[0035] The specific method is as follows:

[0036] (1) Myocardial infarction surgery in mice

[0037] In a laboratory environment, 1% avertin was injected intraperitoneally into C57BL / 6 mice weighing 20-25 grams to achieve general anesthesia. After anesthesia, the mice were depilated and fixed on the operating table, and the endotracheal tube was connected to the ventilator to maintain stable breathing during the operation. Before the operation, all surgical instruments were strictly sterilized to prevent infection. After the skin was cut, two layers of muscle tissue were peeled off at the 2nd and 3rd ribs to expose the heart. After the heart was exposed, the pericardium was torn and a 7-0 suture was used to perform a fixed-point ligation 2-3 mm below the auricle to simulate the conditions of myocardial infarction. After the ligation was completed, the ribs and the muscles above them were repositioned, and the skin was sutured to complete the operation. For the mice in the sham operation group, except for the ligation, the remaining surgical steps were the same as those in the experimental group to ensure the fairness and accuracy of the experiment.

[0038] (2) Immunofluorescence experiment

[0039] When performing immunofluorescence staining analysis of cardiac tissue, first, the frozen sections made of cardiac tissue were fixed in 4% paraformaldehyde solution, the sections were blocked with 10% goat serum, and the sections were incubated overnight with anti-YBX1 antibody (dilution ratio of 1:300) to ensure that the antibody can fully bind to the target protein YBX1. The next day, the sections were incubated with fluorescently labeled secondary antibodies at room temperature for 1 hour for subsequent immunofluorescence detection. In order to mark the cell nuclei, the sections were incubated with DAPI dye for 10 minutes. Finally, an Olympus CKX53 fluorescence microscope was used for observation and analysis. Through the excitation light of a specific wavelength, the immunofluorescence signal of YBX1 protein, the vascular endothelial cells marked by CD31 protein, and the cell nuclei marked by DAPI can be observed, so as to qualitatively and quantitatively analyze the expression and distribution of YBX1 in cardiac tissue.

[0040] Example 2: YBX1 promotes angiogenesis of endothelial cells under hypoxic conditions

[0041] After HUVEC cells were placed in a hypoxic incubator for 24 hours, the protective effect of YBX1 on HUVEC cells induced by hypoxia was detected. CCK-8 experiments showed that upregulation and downregulation of YBX1 increased and decreased the activity of vascular endothelial cells, respectively ( Figure 2 AB). EDU experiments showed that upregulation and downregulation of YBX1 increased and decreased the proliferation capacity of endothelial cells, respectively ( Figure 2 C). Angiogenesis assay showed that up-regulation and down-regulation of YBX1 increased and decreased, respectively, the ability of endothelial cells to form tubular structures ( Figure 2 D).

[0042] The specific method is as follows:

[0043] (1) Cell transfection experiment

[0044] HUVEC cells were grown at 2×10 5The cells were inoculated in a 6-well plate at a density of 100 μg / mL and cultured in a 37°C incubator for 24 hours. After the cells were observed to be in good growth condition, transfection was started. First, the cell culture medium was replaced with serum-free and dual-antibody-free DMEM culture medium. Then, the transfection system was prepared: oligomers or plasmids were diluted in an appropriate amount of Opti-MEM low serum culture medium and gently mixed with a sterile pipette tip; at the same time, lipo-2000 Transfection Reagen was mixed with an appropriate amount of Opti-MEM low serum culture medium, and then gently mixed and placed at room temperature for 5 minutes. Finally, the diluted siRNA or plasmid and the diluted lipo-2000 Transfection Reagen were mixed and placed at room temperature for 15 minutes to form a stable polymer, and then added to the culture plate, and the culture plate was gently shaken to mix it thoroughly, and then placed in a 37°C, 5% CO2 incubator for 8 hours, and then treated with hypoxia. YBX1-specific siRNA (siYBX1) and negative control siRNA (NC) were synthesized by Guangzhou Ruibo Company with a final concentration of 100nM. The targeting sequence of siYBX1 is GGTGCAGGAGAGGAAGGTAA (SEQ ID NO: 2). The YBX1 plasmid uses the pcDNA3.1(+) empty vector as a negative control, and the scrambled sequence and empty plasmid are transfected as negative controls. (2) Cell viability assay (CCK8 assay)

[0045] Cells in the logarithmic growth phase were prepared into single-cell suspension by conventional digestion and subculture methods, and the cell density was adjusted to 1×10 5 / ml, inoculated in a 96-well plate, 100 microliters per well, so that each well has approximately 1,000 to 5,000 cells. Place in a CO2 incubator and culture for an appropriate time at 37°C and 5% CO2 to allow the cells to adhere. According to the experimental design, add overexpression YBX1 plasmid or YBX1 small interfering RNA and continue to culture for 48 hours. Add 10 microliters of CCK-8 solution to each culture well containing cells, put the 96-well plate back into the incubator, and continue to culture for 1-4 hours. The specific time is optimized according to the cell type and experimental conditions. Determine absorbance: Take the 96-well plate out of the incubator and use a microplate reader to measure the absorbance at a wavelength of 450nm, and the reference wavelength is set to 630nm.

[0046] Data analysis: Cell viability was calculated based on the absorbance value. Untreated cells were usually used as the control group, and the cell survival rate of each treatment group was calculated: cell survival rate (%) = (absorbance of treatment group - absorbance of blank group) / (absorbance of control group - absorbance of blank group) × 100%.

[0047] (3) EdU cell proliferation assay

[0048] After treating the cells according to the experimental requirements, dilute EdU to 20μM with complete medium, then add it to the cells to make the final concentration 10μM, and continue to incubate at 37℃ for 2 hours. Remove the EdU working solution, wash the cells 1-2 times with PBS, 3 minutes each time; add 50μL 4% paraformaldehyde to each well to fix the cells at room temperature for 30 minutes; after removing the fixative, wash with PBS 3 times, 3-5 minutes each time; add 100μL permeabilization solution (0.5% TritonX-100 in PBS) to each well and incubate at room temperature for 10-15 minutes; remove the permeabilization solution and wash again with PBS 1-2 times, 3-5 minutes each time. Prepare the reaction solution according to the instructions of the kit, add 50μL of reaction solution to each well, and incubate at room temperature in the dark for 30 minutes; after removing the reaction solution, wash with PBS 3 times, 3-5 minutes each time. Dilute DAPI to 1× with deionized water, add 100 μL of diluted DAPI to each well, and stain for 10 minutes at room temperature in the dark; remove DAPI and wash 3 times with PBS, 3 to 5 minutes each time. Observe EdU-labeled and unlabeled cells under a fluorescence microscope, count and take pictures, and use Image J software for image data analysis.

[0049] (4) Angiogenesis assay

[0050] For the angiogenesis assay, place Matrigel (BD Biosciences) on ice and mix the Matrigel using a pre-cooled pipette tip to avoid creating bubbles. While working on ice, add 50-70 μL of Matrigel to each well. Gently shake the plate to evenly distribute the Matrigel. Place the plate in a 37°C incubator for 30 minutes to allow the Matrigel to solidify, adjust the cell density to the appropriate concentration, add 500 μL of cell suspension to each well, and return the plate to the 37°C incubator. Use an inverted microscope to photograph the cells and tubular structures in each well. Use Image J image analysis software to quantify the total length of the tubular structures.

[0051] Example 3: Overexpression of YBX1 improves cardiac function and promotes angiogenesis in mice with myocardial infarction

[0052] Adeno-associated virus 9 was used to construct a YBX1 overexpression plasmid vector (AAV9-YBX1), which was injected into mice through the tail vein to overexpress the YBX1 gene. The mice underwent MI surgery 3 weeks later, and the cardiac function and angiogenesis of the mice were tested 4 weeks later. The results showed that there was no significant difference in cardiac function between the negative control (NC) group and the YBX1 group in the sham group ( Figure 3 A), but compared with the MI-NC group, the cardiac function parameters such as left ventricular ejection fraction (EF%), fractional shortening (FS%), left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs) were significantly improved in the MI-YBX1 group ( Figure 3 B). By Masson trichrome staining analysis, all mice with myocardial infarction developed scar formation 28 days after myocardial infarction, but the scar size in the MI-YBX1 group was significantly smaller than that in the MI-NC group ( Figure 3 CD). These data further indicate that YBX1 overexpression not only improves left ventricular function but also prevents progressive deterioration of left ventricular remodeling. CD31 staining showed that microvessel density increased in the MI-YBX1 group, suggesting that YBX1 overexpression promotes angiogenesis after MI ( Figure 3 EF).

[0053] The specific method is as follows:

[0054] (1) Construction and infection of AAV9-YBX1 viral vector

[0055] Adeno-associated virus type 9 (AAV9) vector was used to construct a YBX1 overexpression model in vivo. AAV9 vector carrying YBX1 gene and enhanced GFP gene was constructed for overexpression of YBX1 gene. AAV9 virus carrying GFP gene for overexpression of YBX1 was constructed, and AAV9-YBX1 (1.3×10 11 ), and follow-up experiments were performed 3 weeks after administration.

[0056] (2) Myocardial infarction surgery in mice, the method is the same as step (1) in Example 1.

[0057] (3) Ultrasound detection of mouse heart function

[0058] All mice underwent cardiac function assessment 28 days after LAD ligation. Mice were anesthetized with 2-4% isoflurane, depilated, and the coupling agent was covered on the probe ultrasound area. When the heart rate stabilized to 400-500 beats per minute, the VINNO D700LAB high-resolution ultrasound imaging system was used for echocardiography measurement, M-mode recording, and the following cardiac parameters were recorded: ejection fraction (EF%), short-axis shortening (FS%), left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs).

[0059] (4) Masson staining

[0060] Dewax paraffin sections to water: xylene I for 5 minutes, xylene II for 5 minutes, xylene III for 5 minutes, anhydrous ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, rinse with tap water for a few seconds, and wash with tap water and distilled water in turn. Nuclear staining: Use hematoxylin staining solution to stain the nucleus for 5-10 minutes, differentiate with 1% hydrochloric acid alcohol for 5-15 seconds, and wash thoroughly with water; add 0.1-1% lithium carbonate for 5 minutes to increase the degree of anti-blueing, and wash again with water. Use Masson Ponceau acid red solution for 5-10 minutes, wash with 2% glacial acetic acid aqueous solution for 1 minute; then differentiate with 1% phosphomolybdic acid aqueous solution for 3-5 minutes, and wash with 2% glacial acetic acid aqueous solution for 1 minute. For collagen fiber staining, the samples were directly stained with aniline blue aqueous solution or 1% light green aqueous solution for 1-2 minutes without washing, washed with 0.2% glacial acetic acid aqueous solution for 1 minute, dehydrated with 95% ethanol for 2-3 seconds, dehydrated with anhydrous ethanol three times for 5 seconds each time, transparentized with xylene three times for 1 minute each time, and sealed with neutral gum. The fibrosis area was calculated as the total fibrosis area ÷ the left ventricular area, and the image data were processed using Image J software.

[0061] Example 4: Inhibition of YBX1 expression severely impairs cardiac function and inhibits angiogenesis in mice with myocardial infarction

[0062] Adeno-associated virus 9 was used to construct a YBX1 knockdown plasmid vector (AAV9-SH-YBX1), which was injected into mice through the tail vein to inhibit YBX1 gene expression. The mice underwent MI surgery 3 weeks later, and the cardiac function and angiogenesis of the mice were tested after 4 weeks. The results showed that there was no significant difference in cardiac function between the negative control (NC) group and the SH-YBX1 group in the sham group ( Figure 4 A), but compared with the MI+NC group, the cardiac function parameters such as left ventricular ejection fraction (EF%), fractional shortening (FS%), left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs) were significantly inhibited in the MI+SH-YBX1 group ( Figure 4 B). Masson trichrome staining analysis showed that all mice with myocardial infarction developed scar formation 28 days after myocardial infarction, but the scar size in the MI+SH-YBX1 group was significantly larger than that in the MI-NC group ( Figure 4 CD). These data further indicate that inhibition of YBX1 expression not only aggravates left ventricular functional damage, but also leads to progressive deterioration of left ventricular remodeling. CD31 staining showed that microvessel density decreased in the MI+SH-YBX1 group, suggesting that inhibition of YBX1 expression inhibits angiogenesis after MI ( Figure 4 EF).

[0063] The specific method is as follows:

[0064] (1) Construction and infection of AAV9-SH-YBX1 viral vector

[0065] Adeno-associated virus type 9 vector (AAV9) was used to construct a YBX1 knockdown model in vivo. shRNA carrying SH-YBX1 was constructed to inhibit the YBX1 gene; AAV9-SH-YBX1 (1.3×10 11 ), and follow-up experiments were performed 3 weeks after administration.

[0066] (2) Myocardial infarction surgery in mice, the method is the same as step (1) in Example 1.

[0067] (3) Ultrasonic detection of mouse cardiac function, using the same method as step (3) in Example 3.

[0068] (4) The area of ​​myocardial fibrosis was detected by Masson staining, in the same manner as step (4) in Example 3.

[0069] (5) Statistical data analysis

[0070] The experimental data were calculated as mean ± standard error (mean ± SEM), and GraphPad Prism 8.3 software was used for statistical analysis and processing. The two-tailed t-test was used to compare the differences between the two groups, and the one-way analysis of variance (ANOVA) and post hoc Tukey test were used to compare the differences between multiple groups. *P<0.05 was considered statistically significant.

[0071] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of YBX1 as a biomarker for diagnosing myocardial infarction or vascular injury. The amino acid sequence of YBX1 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: Inhibiting the expression of YBX1 can aggravate the damage caused by myocardial infarction; overexpressing YBX1 in vivo can inhibit the heart damage caused by myocardial infarction, thereby protecting the heart.

3. A diagnostic kit for myocardial infarction and vascular injury, comprising a reagent for detecting the expression of YBX1, wherein the amino acid sequence of YBX1 is shown in SEQ ID NO:

1.

4. Use of YBX1 in the preparation of a drug for preventing or treating myocardial infarction and vascular injury, wherein the amino acid sequence of YBX1 is shown in SEQ ID NO:

1.

5. The use according to claim 4, characterized in that: The drug promotes overexpression of YBX1 in vivo, and overexpression of YBX1 promotes the viability and angiogenesis of human umbilical vein endothelial cells.

6. Use of a reagent for detecting the expression of YBX1 in the preparation of a drug for preventing or treating myocardial infarction and vascular injury, wherein the amino acid sequence of YBX1 is shown in SEQ ID NO:

1.

7. Use of a YBX1 activator in the preparation of a drug for preventing or treating myocardial infarction and vascular injury, wherein the amino acid sequence of the YBX1 is shown in SEQ ID NO:

1.

8. A YBX1-specific siRNA molecule, whose targeting sequence is shown in SEQ ID NO:

2.

9. A YBX1 overexpression plasmid vector, which carries the overexpressed YBX1 gene, and the vector used is an adeno-associated virus type 9 vector.

10. Use of the YBX1-specific siRNA molecule according to claim 8 or the YBX1 overexpression plasmid vector according to claim 9 in the preparation of a drug for preventing or treating myocardial infarction and vascular injury.

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