CN115245567B

CN115245567B - Application of FGL1 inhibitor in preparation of medicines for preventing and treating myocardial ischemia injury

Info

Publication number: CN115245567B
Application number: CN202111512864.8A
Authority: CN
Inventors: 齐炼文; 刘群; 刘席花; 尹小建
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-12-11
Filing date: 2021-12-11
Publication date: 2023-06-06
Anticipated expiration: 2041-12-11
Also published as: CN115245567A

Abstract

The invention discloses application of an FGL1 inhibitor in preparation of a medicament for preventing and treating myocardial ischemia injury. The invention discovers that inhibiting the expression of FGL1 gene and reducing the content level of FGL1 protein has the function of preventing and treating myocardial ischemia injury, so that the FGL1 gene inhibitor or the FGL1 protein inhibitor has the prospect of being developed into a medicament for preventing and treating myocardial ischemia injury.

Description

Application of FGL1 inhibitor in preparation of medicines for preventing and treating myocardial ischemia injury

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of an FGL1 inhibitor in preparation of a medicine for preventing and treating myocardial ischemia injury.

Background

Fibrinogen-like protein 1 (FGL 1), also known as hepatocyte-derived fibrinogen-related protein 1 (HFREP-1) or Heparin (HPS), belongs to a member of fibrinogen family, is a specific hepatocyte mitotic active factor, and plays a role in protecting liver and promoting mitosis and proliferation of hepatocytes. FGL1 is located on chromosome 8p22-p21.3, the length of cDNA of gene is 936bp, the gene can code 312 amino acids, N end 1-22 amino acids are signal peptide sequence, and the biological activity of the gene is exerted in homodimer form.

FGL1 has now been found to play a critical role in liver regeneration, cell proliferation, glycolipid metabolism and immune activation. FGL1 as a liver protecting factor accelerates cell growth by promoting mitochondrial mitosis in liver injury, and participates in the process of self-repair of injured liver. FGL1 levels are significantly elevated when the liver is exposed to chemicals, radiation, or hyperglycemic crises, a process that may depend on transcription factor signaling in the FGL1 gene promoter in combination with transcription activator (STAT 3) and long-chain non-coding RNA hepatocyte nuclear factor 1 (HNF 1). Furthermore, FGL1 binds to membrane-specific receptors of hepatocytes, inducing cell proliferation through an autocrine mechanism. Meanwhile, FGL1 is identified as an atypical biomarker of non-alcoholic fatty liver, type 2 diabetes and obesity, involved in regulating adipogenesis, gluconeogenesis and insulin resistance through a variety of molecular mechanisms. FGL1 is a communication factor that signals liver metabolic disorders to other tissues, and participates in liver lipid metabolism in a manner dependent on extracellular regulated protein kinase 1/2 (Erk 1/2), while affecting insulin resistance associated with obesity in adipose tissue and skeletal muscle. Early studies on anti-tumor effects of FGL1 have focused on the field of hepatocellular carcinoma, and have shown that specific silencing of FGL1 inhibits the development of hepatocellular carcinoma through a protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. FGL1 was then significantly upregulated in gastric, non-small cell lung and breast cancers, and silencing in the relevant cancer cell lines inhibited cell proliferation, confirming it to be a tumor suppressor. Further research shows that FGL1 is a novel lymphocyte activating gene 3 (LAG 3) functional ligand, and tumor cells can activate T cell LAG-3 receptor through FGL1, inhibit T cells and realize immune escape, thereby providing a significant direction for tumor immunotherapy.

At present, no correlation report of FGL1 and myocardial ischemia injury exists, and no application of FGL1 inhibitor in preventing and treating myocardial ischemia injury exists. The invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of an FGL1 inhibitor in preparing medicines for preventing and treating myocardial ischemia injury.

The above object of the present invention is achieved by the following technical scheme:

application of FGL1 gene inhibitor or FGL1 protein inhibitor in preparing medicines for preventing and treating myocardial ischemia injury.

Further, the FGL1 gene inhibitor or FGL1 protein inhibitor includes a substance that inhibits FGL1 gene expression or reduces FGL1 protein content level.

Still further, the FGL1 gene inhibitor comprises shRNA or siRNA that interfere with FGL1 gene expression.

Still further, the myocardial ischemia injury includes myocardial ischemia injury caused by myocardial infarction.

A medicine for preventing and treating myocardial ischemia injury comprises FGL1 gene inhibitor or FGL1 protein inhibitor as active ingredient.

The beneficial effects are that:

the invention discovers that inhibiting the expression of FGL1 gene and reducing the content level of FGL1 protein has the function of preventing and treating myocardial ischemia injury, so that the FGL1 gene inhibitor or the FGL1 protein inhibitor has the prospect of being developed into a medicament for preventing and treating myocardial ischemia injury.

Drawings

FIG. 1 shows the FGL1 protein expression level (1A; 1B) and FGL1 mRNA level (1C) in myocardial tissue of myocardial ischemia damaged mice, FGL1 protein expression level (1D) and mRNA level (1E) after primary glycoxin deprivation in heart muscle cells of milk rats.

Fig. 2: AAV-FGL1 shRNA interference prognosis FGL1 protein expression level in mouse myocardial tissue (2A), FGL1 mRNA level in primary myocardial cells of rats transfected with FGL1siRNA (2B);

FIG. 3 echocardiography after myocardial ischemia mouse AAV-FGL1 shRNA intervention (3A), mouse left ventricular ejection fraction, left ventricular foreshortening fraction, left ventricular end-systole inner diameter, and left ventricular end-systole volume level (3B);

FIG. 4 shows the mRNA levels of Nappb, nappa, myh, myh7 (4E) in myocardial primary cells of milk rats, brdU (4A), LDH (4B), CK-MB (4C), bax and Bcl-2 (4D) protein levels after FGL1siRNA stem prognosis.

Detailed Description

The following describes the essential aspects of the invention in detail with reference to the drawings and examples, but is not intended to limit the scope of the invention.

1. Experimental materials

1. Experimental animal

C57BL/6 male mice are bred in cages at 10-12 weeks of age, are kept at constant temperature (23-25 ℃) and are kept at constant humidity (55-70%); milk rats were purchased from Shanghai Bi Kai medical science and technology Co., ltd.1-3 days old.

2. Apparatus and device

Mouse vein visual tail injection fixer (North)Life of Beijing center department); FRESCO17 high speed refrigerated centrifuge (us ThermoFisher Scientific); biosafety cabinet (us ThermoFisher Scientific); synergy 2 multifunctional enzyme labeling apparatus (BioTek company, usa); thermomixer comfort metal bath heater (eppendorf, germany); orbital Shaker TS-10 decolorizing shaker (Kylin-Bell Lab Instraments, haimen Co., ltd.); PROTEANN-II

XI Cell type vertical electrophoresis tank (BIO-RAD Co., U.S.); mini PROTEANII (BIO-RAD Co., USA); powerPac Basic electrophoresis apparatus (BIO-RAD Co., U.S.); a Tanon-520 day gel imaging system (Shanghai Techno Co., ltd.); />

3100LT ultra-high resolution small animal ultrasound imaging systems are available from FUJIFILM VisualSonics company.

3. Apparatus and device

Adeno-associated virus 9 (AAV 9) encapsulates negative control shRNA (negative controlshRNA, NC shRNA) and FGL1 shRNA commissioned Shanghai Jiman Biotechnology Co., ltd, designed and synthesized and encapsulated to a viral titer of 10 ^{^13} Is a virus liquid. Negative control siRNA (NC siRNA) and FGL1siRNA delegated the design and synthesis of shanghai Ji Ma gene pharmaceutical technologies, inc. RIPA strong lysate was purchased from Biyundian (Jiangsu Haomen, china); 30% (w/v) acrylamide/methylene bisacrylamide solution was purchased from a manufacturer (Shanghai, china); 0.5MpH =6.8 Tris-HCl buffer solution and 1.5M ph=8.8 Tris-HCl buffer solution purchased from a growing organism (Jiangsu nanjing, china); cocktail protease inhibitors and phosphatase inhibitors were purchased from Roche, usa; protein loading markers, available from us ThermoFisher Scientific company; mouse creatine kinase isozyme MB (CK-MB) enzyme-linked immunosorbent assay kit and mouse Lactate Dehydrogenase (LDH) enzyme-linked immunosorbent assay kit are purchased from Wuhan Huamei bioengineering Co., ltd; brdU Cell ProliferationAssay Kit is purchased from Cell Signaling in the united states; antibodies Bax, bcl-2, beta-Tubulin were purchased from Wohan Sanying biotechnology Co.

2. Experimental method

1. Establishment and grouping of mouse myocardial ischemia models

A myocardial ischemia model of the C57BL/6 mouse is established by adopting a method of opening and ligating anterior descending branches of left coronary artery (ischemia 24 h).

Three groups were classified into Sham surgery (Sham), ischemia model (AMI), ischemia model+myocardial specific FGL1 silencing (ami+aav-FGL 1 shRNA).

The tail of the mouse is injected with virus liquid intravenously, and a myocardial ischemia model is established after 1 month: the mice are anesthetized by pentobarbital sodium, the mice are fixed on an operating table, the neck epidermis is cut off, the tracheal plug of a breathing machine is inserted into the trachea of the mice, after the breathing frequency of the mice is consistent with the breathing frequency of the breathing machine, the chest epidermis is cut off, muscles are stripped, a small opening is formed at the second rib and the third rib of the left chest by using a blunt surgical instrument, after the position of the heart is observed, the left anterior descending branch of the coronary artery is ligated by using a suture needle with a thread, and the heart is detected after 24 hours.

2. Establishment and grouping of milk rat myocardial primary cell (NRCMs) glycoxygen deprivation (OGD) models

NRCMs cells were anoxic (1% oxygen; 5% carbon dioxide) in an anoxic tank for 6 hours to construct a model of myocardial ischemia injury of NRCMs cells. The groups are classified into a Control group, an OGD group, and an OGD+FGL1 siRNA group 3.

Extraction of NRCMs: wiping the whole body of a milk rat born for 1-3 days with alcohol, cutting the chest cavity (between the first rib and the second rib which are clear and visible from top to bottom) of the mouse, clamping the heart out by forceps, putting the heart into a dish containing PBS, and then cutting the heart into uniform small pieces and putting the uniform small pieces into another dish; washing with PBS, transferring to digestion bottle, adding collagenase, shaking in 37 deg.C water bath, digesting cells, adding serum-containing culture medium into the digestion solution, stopping digestion, centrifuging after digestion of all myocardial tissue, 1000r,5min, removing supernatant, adding erythrocyte lysate into the precipitate, lysing for 2min, diluting lysate with PBS, reversing upside down, centrifuging, discarding supernatant, adding culture medium, re-suspending at 37deg.C, and 5% CO ₂ Incubating in a cell incubator for 2 hours; the fibroblast is attached, and the myocardial cell is in suspension, and the fibroblast is attachedSucking the laminar cell suspension (containing myocardial cells) through a 75 mu M cell sieve, separating to obtain primary myocardial cells of the milk rat, adding a proper amount of culture medium into the separated myocardial cells according to the experimental demand density, adding a fibroblast inhibitor BrdU solution (final concentration of 100 mu M), plating, and culturing; after 24h, the cell plates were gently tapped, the medium was discarded, PBS was washed to replace the new medium, and culture was continued.

Model construction: taking out the cells, discarding the culture medium, washing with PBS once, adding sugar-free and serum-free culture medium, and adding into the culture medium at 1%O ₂ Is anoxic for 6 hours under the condition of (2).

Cell transfection (in the example of 6 well plate required): (1) preparing a transfection solution, namely: 150. Mu.L/well of Opti-MEM medium+FGL1siRNA 3. Mu.g/well; the control group is 150 mu L/well of Opti-MEM culture medium and 3 mu g/well of NC siRNA; and (2) liquid B: opti-MEM Medium 150. Mu.L/well+Lipofectamine ^TM RNAiMAX 12. Mu.L/well; mixing the solution A and the solution B, gently reversing and uniformly mixing, and standing at room temperature for 5min for use; (2) discarding the cell culture medium, washing with PBS buffer solution, and adding 1750 mu L of serum-free double-antibody-free DMEM medium into each well; each well is added with 250 mu L of transfection solution, and the 6-well plate is gently shaken and then placed in an incubator for incubation; (3) after transfection for 6h, the solution is changed, after the solution is washed by PBS buffer solution, 2mL of DMEM high-sugar double-antibody-containing medium with 10% FBS is added into each hole, and the subsequent experiment can be carried out after the culture is carried out overnight.

3. FGL1 gene expression level detection (real-time fluorescence quantitative PCR)

(1) Extraction of RNA: taking out the cells, discarding the culture solution, washing with PBS once, adding a proper amount of Total RNAExtraction Reagent, covering and repeatedly blowing the lysed cells, and standing at room temperature for 5min to completely dissociate the nucleoprotein. Chloroform was added to the lysate, and after vigorous shaking, the mixture was allowed to stand at room temperature for 2min. Centrifuge at 12000rpm for 15min at 4 ℃. The supernatant was gently aspirated and placed in a fresh centrifuge tube, an equal volume of isopropanol was added, and after inversion mixing, the tube was left at room temperature for 10min. Centrifuging at 4deg.C and 12000rpm for 10min, removing supernatant, adding 75% ethanol, washing thoroughly, centrifuging at 4deg.C and 12000rpm for 3min, removing supernatant, drying at room temperature, and dissolving with appropriate amount of DEPC water.

(2) Reverse transcription: removing residual genomic DNA inPreparing a mixed solution in an RNase free centrifuge tube, wherein the components are RNase free ddH ₂ O,5, xgDNAdigester Mix, template RNA was gently pipetted and incubated at 42℃for 2min.

Preparing a reverse transcription reaction system, uniformly mixing the reaction solution in the first step with 4XHifair III SuperMix plus, and setting a reverse transcription program as follows:

(3) Q-PCR quantification: mixing HieffqPCR SYBR Green Master Mix with the target gene front and back primers and template DNA on ice, and carrying out sterile ultrapure water uniformly, and amplifying DNA fragments on a Bio-Rad fluorescent quantitative enzyme-labeling instrument according to a two-step method:

primer sequence:

4. FGL1 protein expression level detection

4.1 detection of FGL1 protein expression level in vivo assay

(1) Protein extraction: injecting 10mg/mL sodium pentobarbital into abdominal cavity according to the dosage of 10mL/kg, anaesthetizing the mice, dissecting the mice, taking out the heart, squeezing the blood in physiological saline, wrapping with tinfoil paper, writing into groups, quick-freezing in liquid nitrogen, placing in liquid nitrogen, and transferring to a refrigerator at-80 ℃ for preservation. Tissue (about 20 mg) at 1/3 of the apex of the heart was excised in a 2mL centrifuge tube. 3 steel balls and 900mL of RIPA strong lysate are added to each tube, and the mixture is subjected to 75Hz cracking for 45s in a tissue grinder. The tissue homogenate was transferred to a 1.5mL centrifuge tube and lysed on ice for 30min. Centrifugation was performed at 13000rpm for 15min at 4℃to allow tissue fragments to be thoroughly centrifuged to the bottom of the tube. Protein supernatants were transferred to new EP tubes and protein concentrations were determined by BCA and 200. Mu.L of protein supernatant was used for the experiment. The other protein supernatant is packaged and stored in a refrigerator at the temperature of minus 80 ℃ for standby.

(2) Protein concentration was determined according to BCA method and protein loading was calculated: (1) preparing a standard protein solution and a working solution: 1.2mL of protein standard preparation solution is added into the protein standard substance, and 25mg/mL of standard protein mother solution is prepared after vortex mixing, and the protein standard substance is stored in a refrigerator at the temperature of minus 20 ℃ for standby. Taking out the standard protein mother solution before use, and diluting the standard protein mother solution into 0.5mg/mL of standard protein working solution by using PBS buffer solution; working solution: preparing working solution from solution A and solution B in the BCA kit according to the volume ratio of 50:1, and preparing the working solution according to the required quantity in situ; (2) preparing a standard curve: 0,1,2,4,8, 12, 16 and 20. Mu.L of the prepared standard protein working solution were added to 96-well plates, respectively, and made up to 20. Mu.L with PBS buffer. Repeatedly preparing two standard curves; (3) sample adding: 1 μl of protein sample solution and 19 μl of PBS buffer solution were added to each well, and two replicates were set for each protein sample; adding 200 mu L BCA working solution into the standard solution hole and the protein sample hole respectively, and incubating for 30min in an incubator at 37 ℃; (4) after the incubation, the absorbance was measured at 562nm in a microplate reader. Calculating the sample concentration of each protein according to a protein standard curve, and calculating the loading volume according to the loading amount of 75 mug; (5) protein denaturation: adding 6×loading buffer (such as 200 μl of protein solution 40 μl of 6×loading buffer) into protein solution, centrifuging, heating in metal bath at 99deg.C for 10min to denature protein, cooling to room temperature, and storing in refrigerator at-20deg.C.

(3) Western blot experiment: (1) fixing clean thick glass plate and thin glass plate on a glue-preparing bracket, preparing separating glue with different concentrations according to the required target protein molecular weight, uniformly adding the separating glue between the two plates by using a liquid-transferring gun, and rapidly adding ddH ₂ O seals the hydraulic pressure flat. After the separating gel is solidified, ddH of the upper layer is added ₂ The O was poured off and the excess water was blotted with a piece of paper. Adding the prepared concentrated gel into the upper layer of the separating gel, and rapidly inserting into sample application hole comb. After the concentrated glue is solidified, taking out and storing in a refrigerator at 4 ℃ for standby; (2) taking out the prepared glueCarefully pull the sample application well comb out, place the device in the electrophoresis tank and add 1 Xelectrophoresis buffer. The samples were thawed and centrifuged by vortexing for use. Sequentially adding samples into the comb holes according to the sample loading volume, and respectively adding 2 mu L of protein markers at the left side and the right side of each plate; after the power was turned on, the voltage was set at 80V and after about 30 minutes the protein sample was flattened into a straight line. After the voltage is switched to 120V, continuing electrophoresis until the protein sample runs to the bottommost part of the gel plate, and stopping electrophoresis; (3) pre-preparing 1 Xwet transfer solution with ddH ratio ₂ O: methanol 10 x wet transfer = 7:2:1. After electrophoresis, the power supply is turned off, a proper amount of 1X wet transfer liquid is added in the square groove in advance, filter paper and sponge in the transfer film clamp are soaked, the white clamp is paved, and the side with the white clamp faces upwards. The thin plate is pried and separated, the thick plate and the glue are immersed in 1X wet transfer liquid together, the glue is shoveled down carefully, the concentrated glue part is cut off and then is paved on a transfer film clamp on the front surface, the glue is paved by a special shovel to remove bubbles, a Nitrocellulose (NC) film and filter paper are paved in sequence, and the bubbles are removed by a roller each time. After the membrane is clamped, the membrane is placed in a membrane transferring groove, 1X wet transfer liquid is poured into the groove, an ice bag is placed in the groove, and a groove cover is covered. The film transferring groove is moved into a basin filled with cold water, and an ice bag is added into the basin for refrigeration. Switching on a power supply, regulating the voltage to 300V at maximum, setting constant current 350mA for 90min, and finishing film transfer; (4) the 5% defatted high protein milk was prepared with 1 XTBST solution and prepared on-line. Taking out NC membrane after membrane transfer, placing in 5% skimmed milk powder, and sealing on a shaker for 2h; (5) a5% Bovine Serum Albumin (BSA) solution was prepared from a 1 XTBST solution and used as a primary antibody dilution, and the primary antibody was diluted according to different antibody dilution ratios for use. The corresponding bands were cut according to the molecular weight of the desired protein, 2mL of diluted primary antibody was added to each band, and each band was pressed with a laminator. Placing the pressed strips into a rotary drum and rotating in a refrigerator at 4 ℃ for incubation overnight; the primary antibody was recovered (typically 3-5 times for reuse), the strip was placed in a 1 XTBST solution and washed on a shaker for 10min, and repeated 3 times. 5% of defatted high protein milk dilution secondary antibody. After the film pressing is completed, the strip is placed at room temperature for incubation for 2 hours; the strip is taken out and placed in a 1 XTBST solution, placed on a shaking table for washing for 10min, and repeatedly washed for 3 times for development. Will beThe solution A and the solution B in the Tanon developing solution are prepared according to the proportion of 1:1, the strips are taken out from the 1 XTBST solution and put into a developing instrument, 500 mu L of developing solution is uniformly added to each strip, the exposure shooting is carried out, the photo is stored, and the analysis is carried out by Image J analysis software.

4.2 detection of FGL1 protein expression level in vitro assays

(1) Cell total protein extraction: the medium was discarded. 1mL of PBS buffer solution was added to the wall for washing, the washing solution was sucked by a pipette, 100. Mu.L of 1 XLoadingbuffer solution was added to each well, and the cells were scraped sufficiently with a cell scraper and transferred to a 1.5mL centrifuge tube. The total cell proteins are extracted by using a total mammalian tissue protein extraction kit of Beijing Buffalo biotechnology limited company.

(2) Western blotting experiments were performed at 4.1.

5. Method for measuring heart function of mouse by ultrasonic imaging method

The method comprises the steps of removing hairs of a chest of a mouse by using depilatory cream, anaesthetizing the mouse by using isoflurane before detection on the next day, fixing the mouse on a mouse board, coating a large amount of couplant on the chest, lowering a probe to the middle of ribs, moving an X axis and a Y axis of the probe, rotating the probe by 30 degrees to 45 degrees, observing a B ultrasonic image on a screen, when the heart rhythm is normal and stable, the heart opens and closes maximally, the aorta is not blocked, the heart cavity is clear, searching an optimal section, clicking an M ultrasonic shooting button of a machine, and recording the image at the moment. Raw data and pictures were derived by plotting and calculating with the small animal cardiac metadata analysis software VisApp.

6. ELISA method for determining NRCMs function

(1) Shifting each reagent of ELISA kit to room temperature (18-25 ℃) for balancing for at least 30min, and preparing each reagent according to the instruction book for standby; (2) and standard substance holes and sample holes to be measured are respectively arranged during sample adding. Adding 100 mu L of standard substance or sample to be tested into each hole, slightly shaking and mixing, coating a plate, and incubating for 2h at 37 ℃; (3) discarding the liquid, spin-drying without washing; adding 100 mu L of biotin-labeled antibody working solution into each hole, covering a new plate patch, and incubating for 1h at 37 ℃; (4) discarding the liquid in the holes, spin-drying, washing the plate for 3 times, soaking for 2min each time, and spin-drying at 200 mu L/hole; adding 100 mu L of horseradish peroxidase avidin working solution into each hole, covering a new plate patch, and incubating for 1h at 37 ℃; (5) discarding the liquid in the holes, spin-drying, washing the plate for 5min, soaking for 2min each time, and spin-drying at 200 mu L/hole; adding 90 mu L of substrate solution into each hole in sequence, and developing for 15-30min at 37 ℃ in a dark place; (6) adding 50 mu L of stop solution into each hole in sequence to stop the reaction; sequentially measuring the optical density (OD value) of each hole at a wavelength of 450nm by using an enzyme-labeled instrument within 5min after the reaction is terminated; and calculating a regression equation of the standard curve according to the OD value, substituting the OD value of the sample into the equation, and calculating the concentration of the sample.

7. Statistical analysis

Data are expressed as mean ± SEM. Two samples were analyzed for statistical differences by independent sample t-test, and multiple samples were analyzed for statistical differences using one-way anova.

The differences were statistically significant with p <0.05 and p <0.01.

3. Experimental results

In the figure 1, 1A and 1B are respectively a western blotting experiment and a tissue immunohistochemical experiment to represent the FGL1 protein level in myocardial tissue of a myocardial ischemia mouse, and the result shows that FGL1 in heart tissue is obviously increased during myocardial ischemia; 1C is the gene level of FGL1 in myocardial tissue of the ischemia mice, and the result shows that the gene level of FGL1 is also obviously increased during ischemia; 1D and 1E are the protein and gene expression levels of FGL1 in primary myocardial cells of milk rats at the time of glucose deprivation, respectively, and as a result, it can be seen that FGL1 is significantly increased in protein and gene levels at the time of myocardial cell injury.

In FIG. 2, 2A is the inhibition of FGL1 protein expression in mouse myocardial tissue by AAV-FGL1 shRNA, and 2B is the inhibition of FGL1 mRNA level in milk rat myocardial cells by FGL1 siRNA. The result shows that FGL1 in myocardial tissue is obviously increased in protein and gene level when mice are subjected to myocardial injury, and FGL1 shRNA or FGL1siRNA can obviously inhibit the expression of FGL1 in protein and gene level.

In fig. 3, 3A is an echocardiogram of a mouse, and 3B is a left ventricular ejection fraction, a left ventricular foreshortening fraction, a left ventricular end-systole inner diameter, and a left ventricular end-systole volume level of the mouse. From the measurement results, compared with the Sham group, the left ventricular ejection fraction and the left ventricular shortening fraction of the AMI group mice are obviously reduced, the inner diameter of the left ventricular end systole and the volume level of the left ventricular end systole are obviously increased, which proves that the myocardial function of the AMI group mice is obviously damaged, and the modeling is successful; compared with the AMI group, the AMI+AAV-FGL1 shRNA group mice have obviously improved left ventricular ejection fraction and left ventricular shortening fraction, and the inner diameter of the left ventricular end systole and the volume level of the left ventricular end systole are obviously reduced, which proves that the myocardial function of the AMI+AAV-FGL1 shRNA group mice is obviously improved, and the myocardial ischemia injury of the AMI mice can be improved by inhibiting FGL1 expression.

FIG. 4 shows the results of BrdU (4A), LDH (4B), CK-MB (4C), bax and Bcl-2 (4D) content determination in NRCMs. As can be seen from the measurement results, compared with the Control group, the OGD group NRCMs have reduced proliferation activity, increased LDH and CK-MB levels, increased Bax protein expression level and reduced Bcl-2 protein expression level, which indicates that the OGD group NRCMs have reduced proliferation activity, increased cell permeability and increased apoptosis level, and the modeling is successful; compared with the OGD group, the OGD+FGL1 siRNA group has the advantages that the NRCMs proliferation activity is improved, the LDH and CK-MB levels are reduced, the Bax protein expression level is reduced, and the Bcl-2 protein expression level is increased, which indicates that the NRCMs proliferation activity of the OGD+FGL1 siRNA group is improved, the cell permeability is improved, the apoptosis level is reduced, the myocardial cell function is obviously improved, and the damage of glucose oxygen deprivation on the NRCMs can be reduced by inhibiting the FGL1 expression. 4E is the gene level of Nappb, nappa, myh, myh7 in NRCMs. From the measurement results, the Nappb transcription level in the OGD+FGL1 siRNA group was increased and the Nappa, myh6 and Myh7 transcription levels were decreased compared with the OGD group, indicating that the myocardial cell damage of the OGD+FGL1 siRNA group was improved.

In conclusion, the inhibition of the expression level of FGL1 has the effect of preventing and treating myocardial ischemia injury, so that the FGL1 inhibitor has the prospect of being developed into a medicament for preventing and treating myocardial ischemia injury.

The above-described embodiments serve to describe the substance of the present invention in detail, but those skilled in the art should understand that the scope of the present invention should not be limited to this specific embodiment.

Claims

The application of FGL1 gene inhibitor in preparing medicaments for treating myocardial ischemia injury is characterized in that: the FGL1 gene inhibitor is shRNA or siRNA interfering with FGL1 gene expression.
2. The use according to claim 1, characterized in that: the myocardial ischemia injury is myocardial ischemia injury caused by myocardial infarction.