Use of inhibitors of cip in the preparation of a medicament for the treatment of myocardial infarction

Abstract

The application discloses application of a CIP inhibitor in preparation of a medicine for treating myocardial infarction, and the application comprises the following steps: using a reagent for inhibiting CIP gene and / or CIP protein expression in preparation of a medicine for treating myocardial infarction, treating cardiac ischemia-reperfusion injury, reducing oxidative stress injury of myocardial cells, and reducing myocardial cell apoptosis. The application proves for the first time that inhibiting CIP protein can alleviate myocardial injury in a myocardial infarction process, reduce oxidative stress and myocardial cell apoptosis, and promote myocardial cell proliferation, and has great significance for treating ischemia-reperfusion injury caused by myocardial infarction, and provides a new technical selection for clinical treatment of myocardial infarction.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of CIP inhibitors in the preparation of drugs for treating myocardial infarction. Background Technology

[0002] Myocardial infarction, also known as myocardial infarction, refers to ischemic necrosis of the myocardium. It occurs when the blood flow in the coronary arteries is drastically reduced or interrupted due to coronary artery disease, causing severe and persistent acute ischemia in the corresponding myocardium, ultimately leading to ischemic necrosis of the myocardium.

[0003] Myocardial infarction has become a serious cardiovascular and cerebrovascular disease that severely endangers patients' health. Patients with myocardial infarction often present with symptoms such as retrosternal pain. The clinical treatment for myocardial infarction is timely revascularization, i.e., percutaneous coronary intervention, to save ischemic myocardium. However, current research has found that myocardial cells are also damaged during ischemia-reperfusion. Therefore, developing drugs that target ischemia-reperfusion injury is also crucial for the treatment of myocardial infarction. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides the application of CIP inhibitors in the preparation of drugs for treating myocardial infarction.

[0005] The first objective of this invention is to provide the use of reagents that inhibit the expression of the CIP gene and / or CIP protein in the preparation of medicaments for treating myocardial infarction.

[0006] A second objective of this invention is to provide the use of agents that inhibit the expression of the CIP gene and / or CIP protein in the preparation of medicaments for treating myocardial ischemia-reperfusion injury.

[0007] A third objective of this invention is to provide the use of reagents that inhibit the expression of the CIP gene and / or CIP protein in the preparation of drugs that alleviate oxidative stress-induced damage to cardiomyocytes.

[0008] A fourth objective of this invention is to provide the use of reagents that inhibit the expression of the CIP gene and / or CIP protein in the preparation of drugs that reduce cardiomyocyte apoptosis.

[0009] A fifth objective of this invention is to provide a medicament for treating myocardial infarction and / or myocardial ischemia-reperfusion injury.

[0010] To achieve the above objectives, the present invention is implemented through the following solution:

[0011] This invention, through in vivo and in vitro, knockdown and overexpression experiments, demonstrates for the first time that CIP protein plays a completely opposite role in myocardial infarction as it does in heart failure and dilated cardiomyopathy. Specifically, during myocardial ischemia-reperfusion, CIP protein promotes myocardial damage, while inhibiting CIP can play a protective role in this ischemia-reperfusion injury process after myocardial infarction treatment.

[0012] This invention seeks protection for the following:

[0013] Application of reagents that inhibit the expression of the CIP gene and / or CIP protein in the preparation of drugs for the treatment of myocardial infarction.

[0014] Application of reagents that inhibit the expression of the CIP gene and / or CIP protein in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

[0015] Application of reagents that inhibit the expression of CIP gene and / or CIP protein in the preparation of drugs that alleviate oxidative stress-induced damage to cardiomyocytes.

[0016] Application of reagents that inhibit the expression of the CIP gene and / or CIP protein in the preparation of drugs that reduce cardiomyocyte apoptosis.

[0017] Preferably, the reagent inhibits the expression of the CIP gene and / or CIP protein in the myocardium.

[0018] Preferably, the reagent comprises siRNA targeting the CIP gene.

[0019] More preferably, the nucleotide sequence of the target sequence of the siRNA is shown in SEQ ID NO.5.

[0020] More preferably, the siRNA is selected from at least one of siRNA1 and siRNA2; the nucleotide sequence of the sense strand of siRNA1 is shown in SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.7; the nucleotide sequence of the sense strand of siRNA2 is shown in SEQ ID NO.8, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.9.

[0021] More preferably, the siRNA is siRNA1, the nucleotide sequence of its sense strand is shown in SEQ ID NO.6, and the nucleotide sequence of its antisense strand is shown in SEQ ID NO.7.

[0022] A drug for treating myocardial infarction and / or cardiac ischemia-reperfusion injury, comprising siRNA targeting the CIP gene.

[0023] Preferably, the nucleotide sequence of the target sequence of the siRNA is as shown in SEQ ID NO.5.

[0024] More preferably, the siRNA is selected from at least one of siRNA1 and siRNA2; the nucleotide sequence of the sense strand of siRNA1 is shown in SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.7; the nucleotide sequence of the sense strand of siRNA2 is shown in SEQ ID NO.8, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.9.

[0025] More preferably, the siRNA is siRNA1, the nucleotide sequence of its sense strand is shown in SEQ ID NO.6, and the nucleotide sequence of its antisense strand is shown in SEQ ID NO.7.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention is the first to demonstrate that inhibiting CIP protein can alleviate myocardial damage during myocardial infarction, reduce oxidative stress and cardiomyocyte apoptosis, and promote cardiomyocyte proliferation. This is of great significance for the treatment of ischemia-reperfusion injury caused by myocardial infarction and provides a new technical option for the clinical treatment of myocardial infarction. Attached Figure Description

[0028] Figure 1 The expression of CIP in the cardiac ischemia-reperfusion injury model is shown in Figure A. The relative expression of CIP mRNA in the non-infarct zone (NIZ), infarct-adjacent zone (BIZ), and infarct zone (IZ) in the mouse infarction model is shown in Figure B. The relative expression of CIP mRNA in the ischemia-reperfusion injury model (IR) and the sham-operated group is shown in Figure B.

[0029] Figure 2 The expression of CIP during the hypoxia-reoxygenation process of cardiomyocytes is shown in Figure A. Figure A shows the mRNA expression level of CIP at different time points (1h, 3h, 6h, 12h, and 24h) during 6h hypoxia-reoxygenation in primary suckling rat cardiomyocytes. Figure B shows the protein expression level of CIP at different time points (1h, 3h, 6h, 12h, and 24h) during 6h hypoxia-reoxygenation in primary suckling rat cardiomyocytes.

[0030] Figure 3 The study investigated changes in the cell viability of cardiomyocytes with CIP knockdown stimulated by hydrogen peroxide. CIP knockdown can alleviate cardiomyocyte death caused by oxidative stress.

[0031] Figure 4A shows the expression of apoptosis-related proteins in cardiomyocytes with CIP knockdown stimulated by hydrogen peroxide; A shows the protein expression of Bax, Bcl2, and Cleaved capased 3; B shows the expression of AKT's basal protein and phosphorylated protein.

[0032] Figure 5 This is a map of the pDC-MCMV-MCS-CMV-EGFP vector.

[0033] Figure 6 A shows the effect of hydrogen peroxide stimulation on cardiomyocytes overexpressing CIP; B shows the effect of CIP overexpression; C shows the decrease in cardiomyocyte viability after CIP overexpression; and D shows the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 after CIP overexpression under H2O2 damage.

[0034] Figure 7 To investigate the effect of CIP knockout on myocardial ischemia-reperfusion injury; A shows the identification of protein expression results after CIP knockout; B shows the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 in the ischemic area of ​​cardiac tissue under myocardial ischemia-reperfusion injury conditions.

[0035] Figure 8 To eliminate the effect of CIP on protein kinase B (AKT).

[0036] Figure 9 To eliminate the effect of CIP knockout on apoptosis in myocardial ischemia-reperfusion injury; A shows the positive rate of apoptosis in myocardial tissue in the ischemia-reperfusion injury area detected by TUNEL assay, with green fluorescence representing TUNEL-positive myocardial cell nuclei and blue fluorescence representing total myocardial cell nuclei; B shows the statistical results of TUNEL apoptosis detection.

[0037] Figure 10 To illustrate the effect of CIP overexpression on apoptosis in myocardial ischemia-reperfusion injury, green fluorescence represents TUNEL-positive cardiomyocyte nuclei; blue fluorescence represents total cardiomyocyte nuclei; and red fluorescence represents TNNT-positive cardiomyocytes. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0039] Example 1: Downregulation of CIP expression in a myocardial ischemia-reperfusion injury model

[0040] I. Experimental Methods

[0041] 1. Establishment of a model of myocardial ischemia-reperfusion injury (I / R)

[0042] Male rats (C57 / BL6) aged 8–10 weeks were randomly divided into two groups, designated as the surgical group (IR group) and the sham group (sham group). The surgical group was a model of myocardial ischemia-reperfusion injury (I / R), which conforms to the pathological changes of human myocardial infarction. The sham group served as a control.

[0043] Surgical group: The rats were anesthetized with 1.5% sodium pentobarbital via intraperitoneal injection, fixed in a supine position, and a midline incision was made in the neck. A cannula was inserted to maintain normal respiration. The thoracic cavity was opened in the intercostal space where the heartbeat was most pronounced. The ribs were opened using a facial retractor, and the pericardium was torn open. The retractor was then pressed downwards to expel the heart from the thoracic cavity. The heart was gently grasped with the thumb, index, and middle fingers of the left hand. Using the left coronary vein as a landmark below the left atrial appendage, a 5-0 non-invasive suture needle was inserted 2 mm below the root of the left atrial appendage, passing through the myocardial surface and exiting near the pulmonary artery conus. After the heart stabilized, a slipknot was made to ligate the left anterior descending coronary artery. Darkening of the corresponding area on the heart surface indicated successful ischemia. After 45 minutes, the suture was loosened, and the heart surface turned red, indicating myocardial tissue reperfusion. The ribs, muscles, and skin were sutured layer by layer, and the ventilation volume was gradually reduced. After the rat regained spontaneous breathing, the endotracheal tube was removed. At the same time, the trachea was observed for secretions. If there were secretions, they were aspirated with a syringe with a catheter. The tracheal wound was sutured with a 7-0 atraumatic suture needle. The neck muscles and skin were then sutured layer by layer. The rat was then moved from a supine position to a right lateral decubitus position until it regained consciousness.

[0044] The sham surgery group: The procedure was basically the same as the surgical group, except that a 5-0 non-invasive suture needle was used to pass through the myocardium 2 mm below the root of the left atrial appendage, exited next to the pulmonary artery conus, and passed below the left anterior descending coronary artery without ligation.

[0045] 2. Detection of CIP mRNA expression by real-time PCR

[0046] (1)Acquisition of materials

[0047] Both the surgical group and the sham surgery group underwent tissue sampling 24 hours after the surgery, using the following methods:

[0048] Eight weeks post-surgery, mice underwent echocardiography and were anesthetized with 1-2% isoflurane. After anesthesia, a transverse incision was made along the lower sternal border to fully expose the upper abdominal cavity, followed by incision of the diaphragm. The ribs on both sides were then cut to fully expose the heart. The blood vessels at the base of the heart were fixed with forceps and cut off before the heart stopped. The heart was quickly placed in pre-cooled PBS solution. While the heart pumped out the remaining blood from the chambers, the connective tissue surrounding the heart and the left and right atria were removed. The ventricular tissue was weighed, immediately flash-frozen in liquid nitrogen, and then stored at -80°C. After ligation of the left anterior descending artery, the area in the heart without blood supply (whitened) was designated the infarct zone (IZ). The area within approximately 0.5 cm of the IZ was designated the adjacent infarct zone (BIZ), and other areas far from the IZ and unaffected by blood supply were designated the non-infarct zone (NIZ).

[0049] (2) RNA extraction and reverse transcription

[0050] RNA was extracted from ventricular tissue using standard methods.

[0051] Prepare the following solution systems (two 1.5 mL EP tubes): System 1: 5X gDNA Eraser buffer: 2 μL*n (indicating the specific number of EP tubes); gDNA Eraser: 1 μL*n; System 2: Primer script RT Enzyme MIX I: 1 μL*n; RT primer MIX: 1 μL*n; 5X primer script buffer 2: 4 μL*n; RNase free H2O: 4 μL*n.

[0052] Add the calculated PCR water and RNA to the labeled EP tubes, totaling 7 μL. Add 3 μL / well of reaction mixture 1 to the EP tubes containing water and RNA, vortex, and gently centrifuge. Turn on the PCR instrument, place the EP tubes in the middle, tighten the PCR instrument cap, and set the program to: 42℃, 2 min, reaction volume 10 μL. After the reaction, remove the EP tubes, add 10 μL / well of reaction mixture 2, vortex, and centrifuge. Place the tubes back into the PCR instrument, and set the program to: 37℃, 15 min; 85℃, 5 s; 4℃, reaction volume 20 μL. The final product is the cDNA extracted through reverse transcription.

[0053] The obtained cDNA was used as a template for real-time PCR reaction. The primers used are shown in Table 1.

[0054] Table 1 Primers for Real-Time PCR

[0055]

[0056] The real-time PCR reaction system was as follows: TB Green premix Ex Taq II, 10 μL; cDNA, 0.2 μL; nuclease-free water, 7.8 μL; ROX reference Dye, 0.4 μL; upstream primer (10 μM), 0.8 μL; downstream primer (10 μM), 0.8 μL.

[0057] The quantitative PCR reaction program was as follows: 95℃ for 30s; 95℃ for 5s; 60℃ for 30s, followed by plate reading, for a total of 40 cycles. Melting curve analysis was performed at temperatures ranging from 55℃ to 95℃, with readings taken every minute.

[0058] II. Experimental Results

[0059] like Figure 1 As shown in Figures A and B, compared to the sham-operated group, the expression level of CIP protein in the operated rats was significantly decreased during ischemia-reperfusion injury, and the expression level of CIP was even lower in the infarct area than in the non-infarct area. This indicates that under normal ischemia-reperfusion injury conditions, CIP protein plays a role in promoting injury, so the body will correspondingly reduce CIP expression to alleviate this injury.

[0060] Example 2: Downregulation of CIP expression during cardiomyocyte hypoxia-reoxygenation

[0061] I. Experimental Methods

[0062] 1. Isolation and culture of primary neonatal rat cardiomyocytes (NRCM)

[0063] (1) Separation of the heart

[0064] Spray your hands with alcohol, then use large scissors to break the skin and forceps and small scissors to remove the rat's heart. Place the heart in a culture dish containing 1xPBS, remove excess tissue and blood, and transfer it to a culture dish containing 1xPBS on ice for temporary storage.

[0065] (2) Solution preparation

[0066] Prepare 4 tubes of trypsin neutralization solution (i.e., DMEM (45 mL / tube) containing 10% (v / v) domestic serum (manufacturer: Excell, catalog number: FSP500) (5 mL / tube)), and preheat at 37°C after preparation; prepare 2 tubes of primary digestion solution (i.e., solution A, specific formula: NaCl, 137 mmol / L; KCl, 4 mmol / L; NaHCO3, 4.2 mmol / L; Glucose, 5 mmol / L; pH, 7.68; filtered and sterilized) 30 mL / tube + 0.25% trypsin (Trpsin) 15 mL / tube), and preheat 1 tube at 37°C after preparation.

[0067] Prepare 3 tubes of cardiomyocyte culture medium (i.e., 43.5 mL DMEM / tube + 0.5 mL penicillin antibody / tube + 5 mL Transe serum / tube + 0.5 mL sodium pyruvate / tube + 0.5 mL glutamine / tube), and mix by inverting.

[0068] (3) Tissue digestion

[0069] Add 7 mL of 1xPBS to a T50 Erlenmeyer flask. Wipe your index finger clean with sterile paper, spray with alcohol onto the laminar flow hood, and using your index finger as the work surface, place the heart inside. Cut the tissue into four portions with small scissors and place them into the Erlenmeyer flask. Seal the Erlenmeyer flask with aluminum foil (containing a rotor), shake several times, and discard the first non-tissue fluid inside the laminar flow hood. Add 5 mL of preheated primary digestion solution at 37°C to the Erlenmeyer flask, seal with aluminum foil, and digest at 37°C with spiral shaking for 5 minutes outside the laminar flow hood.

[0070] During digestion, prepare 50 mL centrifuge tubes, attach a 70 μm filter to the tube opening, and add an equal volume of trypsin neutralization solution to the digestion solution. While waiting for digestion, gelatin can be used for plating, adding 1 / 2 the volume of each cell well, and incubating at 37°C for at least 30 min.

[0071] When the first tube of primary digestion solution has been used down to 20 mL, the second tube of digestion solution can be preheated at 37°C. Whether to increase the amount of new primary digestion solution or extend the digestion time depends on the turbidity of the previous digestion solution (it usually becomes turbid during the 3rd to 5th digestion). The clearer the solution, the more intense the digestion needs to be, until the digested tissue blocks turn into small, white, snot-like connective tissue particles, at which point the repeated digestion can be stopped. During the digestion process, the number of digestions, the amount of digestion solution added, and the digestion time should be recorded with pen and paper. When transferring the conical flask, the bottom of the flask should be supported with some fingers to prevent it from falling vertically.

[0072] After tissue digestion, alcohol is sprayed into the digester. The first digestion solution is discarded. In subsequent digestions, the digestion solution is transferred to a 70μm filter screen for filtration and neutralization. Fresh primary digestion solution is added to the conical flask to disperse and mix the aggregated particles. This process is repeated until all heart tissue is digested.

[0073] (4) Centrifugation

[0074] After digestion, the filtered and neutralized tissue digest was centrifuged at 1000 rpm for 10 min. During centrifugation, culture plates (6-well plate, 1 mL / well; 12-well plate, 500 μL / well; 24-well plate, 250 μL / well) were incubated with gelatin at 37°C with 5% CO2 for 0.5 h.

[0075] (5) Adherent culture

[0076] Calculate the total volume of cardiomyocyte culture medium needed for equal distribution, i.e., 5 mouse hearts / 10cm culture dish, 10mL / 10cm cell culture dish, to determine the volume of resuspending medium used to precipitate cells. After centrifugation, remove the supernatant from the centrifuge tubes, add cardiomyocyte culture medium to resuspend, collect the cell pellet from multiple centrifuged tubes into one tube, evenly distribute it into 10cm cell culture dishes, and incubate in a cell culture incubator at 5% CO2 and 37°C for 1-2 hours. This process is to allow fibroblasts to adhere pre-adhere to the culture dish, thus distinguishing them from cardiomyocytes.

[0077] (6) Cardiac cell recycling

[0078] After fibroblasts adhered to the walls of a 10cm cell culture dish, the supernatant was aspirated into a 50mL centrifuge tube. The culture dish was washed with fresh cardiomyocyte culture medium, and the medium was then collected back into the centrifuge tube. After washing, the surface of the covered culture dish was observed under a microscope. If there were many residual cardiomyocytes (bright, round cardiomyocytes, and gray, irregularly shaped fibroblasts), the dish was washed several more times. The supernatant and culture medium were combined to obtain the NRCM suspension.

[0079] (7) Cardiac cell culture

[0080] Remove the gelatin-incubated culture plate, discard the gelatin, mix the NRCM suspension, and evenly seed the cells into the cell culture plate at a density of 1.5 newborn rats per six-well plate. Culture the cells in complete medium (high glucose DMEM containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin antibiotics (P / S)) at 37°C and 5% CO2 for 16 h.

[0081] 2. Hypoxia-reoxygenation experiment

[0082] (1) Preparation of hypoxia buffer

[0083] The formulation of myocardial hypoxia buffer is as follows: HEPES buffer 4mM, NaCl 117mM, KCl 12mM, CaCl2 0.9mM, MgCl2 0.49mM, sodium lactate 20mM. The buffer is prepared according to the reagent preparation method. Then, a mixed gas (containing 5% CO2 and 95% N2) is slowly blown through the buffer for 10 minutes to remove dissolved oxygen from the buffer, thus obtaining the hypoxia buffer.

[0084] (2) Hypoxic culture

[0085] The original culture medium of NRCM was removed, and the cells were washed with PBS and replaced with hypoxia buffer. The hypoxic cells were then placed in a hypoxia incubator (i.e., nitrogen gas plus oxygen content of less than 1%) and cultured at 37°C for 6 hours, which was designated as the hypoxia group (i.e., Hypoxia).

[0086] NRCM cultured at 37℃ and 5% CO2 for 6 hours was used as a control and was designated as the normoxic group (i.e., Normoxia).

[0087] (3) Reoxygenation culture

[0088] The hypoxic group was removed from the hypoxic incubator, the hypoxia buffer was removed, and it was replaced with high-glucose DMEM medium containing 10% (v / v) FBS. The medium was then cultured at 37°C and 5% CO2 for 1 h, 3 h, 6 h, 12 h, and 24 h (i.e., R1 h, R3 h, R6 h, R12 h, and R24 h) to induce reoxygenation damage.

[0089] 3. Detection of CIP mRNA expression by quantitative real-time PCR

[0090] Following the method described in Example 1, total RNA was extracted from cells in the normoxic group and the hypoxic groups at R1h, R3h, R6h, R12h, and R24h, and then reverse transcribed to obtain cDNA.

[0091] The cDNA obtained from reverse transcription was used as a template for real-time PCR. The primers used are shown in Table 1. The real-time PCR reaction system and procedure were the same as in Example 1.

[0092] 4. Western blot analysis of CIP protein expression.

[0093] Total protein was extracted from cells in the normoxic group and the hypoxic groups at R1h, R3h, R6h, R12h and R24h, respectively. Protein concentration and protein denaturation were measured and Western blot analysis was performed. Information on the antibodies used is shown in Table 2.

[0094] Table 2. Antibodies used for Western blot detection of CIP

[0095]

[0096] II. Experimental Results

[0097] like Figure 2 As shown in A, compared with the normoxic group, the overall CIP mRNA expression level was reduced in the hypoxic group after reoxygenation; Figure 2 As shown in Figure B, compared with the normoxic group, the overall CIP protein expression was also reduced in the hypoxic group after reoxygenation. This indicates that both CIP RNA and protein levels are reduced under hypoxia-reoxygenation injury.

[0098] The above results indicate that CIP protein is a detrimental protein to the body during ischemia-reperfusion injury.

[0099] Example 3: Knockdown of CIP reduces cardiomyocyte apoptosis

[0100] I. Experimental Methods

[0101] 1. Design of siRNA

[0102] Using the first to fifth bp of the CDS sequence of the CIP gene (Gene ID: 681849) as the target sequence (SEQ ID NO. 5), two CIP-specific siRNAs (i.e., si-CIP-1 and si-2) were designed, with the specific sequences as follows:

[0103] si-CIP-1-Chain of Justice: 5'-GGACCAGAGGAUACUGGAUTT-3'(SEQ ID NO.6);

[0104] si-CIP-1-antisense chain: 5'-AUCCAGUAUCCUCUGGUCCTT-3' (SEQ ID NO.7);

[0105] si-CIP-2-Chain of Justice: 5'-GGCCCAAGUCUCUGGCUAUTT-3'(SEQ ID NO.8);

[0106] si-CIP-2-Ansense Chain: 5'-AUAGCCAGAGACUUGGGCCTT-3' (SEQ ID NO.9).

[0107] *Regarding SEQ ID NO. 6-9 in the sequence listing of this specification: According to the editing rules of WIPOSequence software, the nucleotide sequence must only contain the symbols listed in "WIPOST.26 Annex I Part 1". The base "T" is "U" in the RNA sequence. Therefore, SEQ ID NO. 6-9 in this specification are substantially the same as SEQ ID NO. 6-9 in the sequence listing.

[0108] The universal negative siRNA (i.e., si-NC, manufacturer: Gemma Gene, catalog number: A06001) was used as a control.

[0109] 2. Cell Culture

[0110] H9C2 cardiomyocytes (Wuhan Pronosai Life Science Co., Ltd.) were injected at a rate of 1.0 × 10⁻⁶ cells per milliliter. 5 Cells were seeded at a density of 1000 cells per well in six-well plates and cultured in complete medium (high glucose DMEM containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin antibiotics (P / S)) at 37°C for 16 h at 5% CO2.

[0111] 3. Transfection

[0112] Cell growth was examined under a microscope. When the confluence of H9C2 cardiomyocytes reached 60%–70%, the old culture medium was discarded. The cells were washed twice with PBS corresponding to the size of the culture dish. The corresponding amount of fresh culture medium (serum-free and antibiotic-free) was placed into the culture dish. si-CIP-1–2 and si-NC were transfected into H9C2 cardiomyocytes using RNAiMAX transfection reagent (Invitrogen, catalog number 13778), and the resulting cells were designated as si-CIP-1 group, si-CIP-2 group, and si-NC group, respectively. Six hours after transfection, the old culture medium was replaced with fresh complete culture medium.

[0113] 4. Irritation from hydrogen peroxide (H2O2)

[0114] Forty-eight hours after transfection, H2O2 was added at a working concentration of 290 μM to the si-CIP-1, si-CIP-2, and si-NC groups to obtain the hydrogen peroxide stimulation group (i.e., the H2O2 group); serum-free high-glucose DMEM medium was added to the si-CIP-1, si-CIP-2, and si-NC groups as a control to obtain the control group (i.e., the Ctrl group). Culture was then continued for another 12 hours.

[0115] 5. Cell viability assay (CCK8 cytotoxicity assay)

[0116] After H2O2 treatment, H9C2 cardiomyocytes were washed with serum-free DMEM to remove excess hydrogen peroxide and avoid interfering with CCK-8 detection. 10 μL (96 wells) of CCK-8 solution was added to each well, and the cells were incubated for 2 hours. The absorbance was then measured at 450 nm.

[0117] 6. Western blot analysis of the expression of apoptosis-related proteins.

[0118] (1) Explanation of apoptosis-related indicators

[0119] B-cell lymphoma-2 gene (Bcl2), BCL2-Associated X protein (Bax, Bcl-2 family proteins directly regulate PTP). Among these proteins, the anti-apoptotic protein Bcl-2 can preserve membrane potential and block the release of cytochrome C. Bax, on the other hand, is a pro-apoptotic protein that eliminates mitochondrial membrane potential by affecting PTP, promoting the release of cytochrome C. Therefore, the Bcl-2 / Bax ratio can be used to measure the degree of cellular apoptosis.

[0120] Caspase-3: Caspases can directly disrupt cellular structures, such as cleaving the nuclear lamina. During apoptosis, laminins, as substrates, are cleaved by caspases at a fixed site near the center, leading to laminin disintegration and chromatin condensation. In addition, caspases can inactivate or downregulate enzymes involved in DNA repair, mRNA cleavage proteins, and DNA cross-linking proteins. Due to the action of DNA, the function of these proteins is inhibited, hindering cell proliferation and replication and inducing apoptosis. Caspase-3 is a type of caspase protease, and its increased expression strongly suggests increased apoptosis.

[0121] (2) Detection method

[0122] Total cellular protein was extracted from the hydrogen peroxide-stimulated si-CIP-1 group (i.e., siCIP1+H2O2) and si-NC group (i.e., siNC+H2O2) and the control group si-CIP-1 group (i.e., siCIP1+Ctrl) and si-NC group (i.e., siNC+Ctrl) in cell culture dishes and detected by Western blot according to the method in Example 2. The information of the antibodies used is shown in Table 3.

[0123] Table 3. Antibodies used for Western blot detection of apoptosis-related proteins.

[0124]

[0125] II. Experimental Results

[0126] like Figure 3 As shown, CIP knockdown can alleviate oxidative stress-induced cardiomyocyte death. This indicates that, compared to the control group, cell density increased after hydrogen peroxide stimulation in CIP-knockdown cells, and CIP knockdown alleviated hydrogen peroxide-induced H9C2 cardiomyocyte death.

[0127] like Figure 4 As shown in Figure A, compared with the control group, under hydrogen peroxide stimulation, the expression of the pro-apoptotic protein Bax was reduced in CIP-knockdown H9C2 cardiomyocytes, while the expression of the anti-apoptotic protein Bcl-2 was increased, and the expression of caspase-3 splice variants was also increased; Figure 4 As shown in B, the proportion of phosphorylated AKT increased in the CIP knockdown group under hydrogen peroxide stimulation.

[0128] The above results indicate that knocking down CIP reduces oxidative stress-induced apoptosis.

[0129] Example 4: Overexpression of CIP promotes cardiomyocyte apoptosis

[0130] I. Experimental Methods

[0131] 1. Construction of CIP overexpression vector

[0132] Using mouse heart cDNA as a template, the CIP gene fragment (SEQ ID NO.12) for constructing the overexpression vector was amplified using upstream amplification primer: 5'-ACCTCTCTCTCATTCTTTCACC-3' (SEQ ID NO.10) and downstream amplification primer: 5'-CACTTCCACTCCAGCTTCC-3' (SEQ ID NO.11).

[0133] The CIP gene fragment (SEQ ID NO.12) was inserted into... Figure 5 Between the BamHI and NotI sites of the pDC-MCMV-MCS-CMV-EGFP vector shown (the same as "the adenoviral empty vector harboring the murine cytomegalovirus (mCMV) promoter" in PubMed literature (PMID: 33677093), we obtain vectors that overexpress CIP and vectors that overexpress GFP, named ad-CIP vector and ad-GFP vector.

[0134] 2. Adenovirus packaging

[0135] The ad-CIP vector and pDC-MCMV-MCS-CMV-EGFP vector were sent to Jikai Gene Company for adenovirus packaging, resulting in adenovirus overexpressing CIP (named V-CIP) and control virus (named V-GFP).

[0136] 3. Cell Culture

[0137] H9C2 cardiomyocytes were cultured according to the method described in Example 2.

[0138] 4. Transfection

[0139] Following the method described in Example 2, V-CIP and V-GFP were transfected into H9C2 cardiomyocytes, and the resulting cells were designated as the ad-CIP group and the ad-GFP group, respectively.

[0140] 5. Western blot analysis of CIP protein expression.

[0141] 48 hours after transfection, the expression of CIP in the ad-CIP group and the ad-GFP group was detected by Western blot according to the method in Example 2.

[0142] 6. Irritation from hydrogen peroxide (H2O2)

[0143] Forty-eight hours after transfection, H2O2 was added to the ad-GFP group and the ad-CIP group at a working concentration of 290 μM to obtain the hydrogen peroxide stimulation group (i.e., the H2O2 group); high-glucose serum-free DMEM medium was added to the ad-GFP group and the ad-CIP group as a control to obtain the control group (i.e., the ctrl group). The culture was then continued for another 12 hours.

[0144] 7. Cell viability assay (CCK8 cytotoxicity assay)

[0145] CCK8 detection was performed on the H2O2 group and the ctrl group according to the method in Example 2.

[0146] 8. Western blot analysis of the expression of apoptosis-related proteins.

[0147] Total cellular protein was extracted from the hydrogen peroxide-stimulated ad-CIP group (i.e., ad-CIP+H2O2) and ad-GFP group (i.e., ad-GFP+H2O2), as well as the control group ad-CIP group (i.e., ad-CIP+Ctrl) and ad-GFP group (i.e., ad-GFP+Ctrl). Western blot analysis was performed according to the method described in Example 2, and information on the antibodies used is shown in Table 3 of Example 3.

[0148] II. Experimental Results

[0149] like Figure 6 As shown in A, CIP overexpression was successful. Figure 6 As shown in B, compared with the control group, cardiomyocyte viability decreased after CIP overexpression; Figure 6 As shown in C, compared with the control group, the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 under H2O2 damage was observed by CIP overexpression.

[0150] The above results indicate that overexpression of CIP protein promotes cardiomyocyte apoptosis.

[0151] Example 5: Knocking down CIP reduces apoptosis caused by myocardial ischemia-reperfusion injury.

[0152] I. Experimental Methods

[0153] 1. Establishment of a model of myocardial ischemia-reperfusion injury (I / R)

[0154] Eight to ten-week-old CIP knockout mice (i.e., “CIP-KO mice” in PubMed literature (PMID:26436652)) and WT control mice (weight controlled at 23–28 g, housed in the SPF-grade animal culture room of the First Affiliated Hospital of Sun Yat-sen University) were randomly divided into two groups. The surgical group and sham-operated group were prepared according to the method in Example 1, that is, CIP knockout mice (KO+I / R) in the surgical group, control mice (HET+I / R) in the surgical group, CIP knockout mice (KO+sham) in the sham-operated group, and control mice (HET+sham) in the sham-operated group were obtained.

[0155] 2. Western blot analysis of the expression of apoptosis-related proteins.

[0156] Heart tissue samples were isolated from the four groups of mice 24 hours after the operation, and protein samples were extracted. Western blot analysis was performed according to the method in Example 2. Information on the antibodies used is shown in Table 3 of Example 3.

[0157] 3. TUNEL Apoptosis Detection Kit for Detecting Cell Apoptosis

[0158] (1) Paraffin embedding

[0159] Heart tissue was isolated from four groups of mice 24 hours after surgery, fixed with paraformaldehyde, and sent to the company (Saiwell Biotechnology Co., Ltd.) to prepare paraffin tissue sections.

[0160] (2) Dewaxing

[0161] Place the paraffin tissue sections on a metal baking rack and bake them in an oven at 65°C for 1–2 hours. This makes the sections adhere more firmly and less likely to fall off, and it also facilitates dewaxing.

[0162] In the pathology lab, paraffin sections are sequentially immersed twice in a glass jar containing xylene, for 10 minutes the first time and 5 minutes the second time. The immersion time can be adjusted according to the season and the progress of dewaxing to ensure thorough dewaxing. After dewaxing, the sections are first immersed in 100% anhydrous ethanol for 5 minutes for hydration, and this process is repeated once.

[0163] (3) Immersion

[0164] Immerse the slides in gradient ethanol solutions (90%, 80%, and 70%) once each, for 3–5 minutes each time. After hydration, rinse the slides 2–3 times with PBS, then remove excess water by swishing. If water cannot be removed, carefully wipe with absorbent paper to remove excess water around the sample, being careful not to touch the sample itself. To facilitate subsequent liquid incubation, use a hydrophobic histochemical pen to draw a closed circle along the tissue outline. To prevent the samples from drying out during the experiment, complete the process one by one, placing the circled samples in a humidified chamber containing water.

[0165] (4) Transparent

[0166] The 2 mg / mL proteinase K solution was diluted with PBS to a concentration of 20 μg / mL to obtain the proteinase K dilution. The heart sample was thoroughly soaked with the proteinase K dilution and permeabilized in a humidified chamber at room temperature for 20 min.

[0167] Rinse the sample 2-3 times with PBS solution, gently remove excess liquid, and carefully and gently wipe the liquid around the sample on the slide with absorbent paper, avoiding contact with the heart sample. Place the sample, after blotting off the surrounding moisture, in a humidifier to prevent the heart sample from drying out too much.

[0168] (5) Labeling and TUNEL detection

[0169] The dried heart samples were tested using the TUNEL apoptosis detection kit (Yisheng Biotechnology) according to the instructions. The specific procedures are as follows:

[0170] Dilute an appropriate amount of 5×Equilibration Buffer with deionized water to obtain 1×Equilibration Buffer. Soak the heart tissue thoroughly in 1×Equilibration Buffer for 20 minutes at room temperature. Wash away most of the 1×Equilibration Buffer with absorbent paper, keeping the heart tissue moist. To prevent the liquid from shaking, you can also place a damp paper towel or cotton at the bottom of the box.

[0171] Dissolve the Alexa Fluor 488-12-dUTP Labeling Mix stored at -20℃ on ice. Prepare sufficient TdT solution according to the sample quantity and instructions. Add TdT incubation buffer to the heart sample, carefully place it in a dark, light-proof humidified box, and transfer it to an incubator to incubate at 37℃ for 60 minutes.

[0172] Remove the coverslip and, under light-protected conditions, place the tissue section in a 5cm incubator containing PBS solution. 2Add 50 μL of TdT incubation buffer to the cells, taking care not to let them dry out. After this, protect the slide from light. To ensure even reagent distribution, gently cover the heart tissue with a coverslip. Wash slowly in a staining jar with shaking for 5 minutes, then wash twice more with clean PBS. Remove the PBS solution from the slide surface by swishing, and gently blot away any remaining moisture with absorbent paper.

[0173] Under light-protected conditions, add the prepared DAPI solution to the area of ​​the tissue sample on the glass slide and incubate at room temperature for 6 minutes. After staining the cell nuclei, place the tissue sample in a staining jar containing deionized water and wash for 5 minutes. Change the liquid and wash twice more.

[0174] Shake off any excess water from the slide, apply anti-quenching mounting solution around the sample area, cover with a coverslip, seal the coverslip with nail polish, and store in a light-proof slide box.

[0175] The samples were observed and photographed under fluorescence at 520±20 nm using a fluorescence microscope.

[0176] II. Experimental Results

[0177] like Figure 7 As shown in Figure A, CIP protein was almost not expressed in mice, indicating successful CIP knockout. Figure 7 As shown in B, in CIP knockout mice, the expression levels of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 in the ischemic area of ​​cardiac tissue are reduced under myocardial ischemia-reperfusion injury.

[0178] like Figure 8 As shown, the phosphorylation level of protein kinase B (AKT) was increased in CIP knockout mice, indicating that CIP knockdown activated AKT phosphorylation and promoted cell proliferation.

[0179] like Figure 9 As shown in A and B, the number of YUNEL-positive cells in CIP knockout mice was significantly reduced under myocardial ischemia-reperfusion injury conditions, indicating that the number of apoptotic cells was reduced after ischemia-reperfusion injury surgery in CIP knockout mice.

[0180] The above results indicate that CIP protein is detrimental to the body in ischemia-reperfusion injury, and knocking out CIP reduces cell apoptosis caused by myocardial ischemia-reperfusion injury and alleviates myocardial infarction.

[0181] Example 6: Overexpression of CIP reduces apoptosis induced by myocardial ischemia-reperfusion injury.

[0182] I. Experimental Methods

[0183] 1. Establishment of a model of myocardial ischemia-reperfusion injury (I / R)

[0184] 8–10 week old CIP conditional overexpression mice (i.e., the “CIP” mice in the PubMed literature (PMID:26436652)) KI Mice (weighing 23-28g and housed in the SPF-grade animal culture room of the First Affiliated Hospital of Sun Yat-sen University) were randomly divided into two groups. One group was induced to overexpress crease and CIP with tamoxifen at 8 weeks to obtain CIP-overexpressing mice (cre+ group), and the other group was not treated with tamoxifen to obtain control mice (cre- group).

[0185] The cre+ group and cre- group were treated according to the method of the surgical group in Example 1, thus obtaining the cre+ group and cre- group with myocardial ischemia-reperfusion injury.

[0186] 2. TUNEL Apoptosis Detection Kit for Detecting Cell Apoptosis

[0187] Twenty-four hours after the operation, heart tissues from the cre+ group and the cre- group were separated and subjected to TUNEL cell apoptosis detection according to the method in Example 5.

[0188] We also used an antibody against skeletal muscle fast muscle troponin (TNNT) (manufacturer: proteintech, catalog number: 15513-1-AP) to label cardiomyocytes in cardiac tissues from the cre+ and cre- groups.

[0189] II. Experimental Results

[0190] like Figure 10 As shown, TUNEL staining revealed an increased proportion of apoptosis induced by myocardial ischemia-reperfusion injury in CIP-overexpressing mice compared to control mice. This indicates that CIP overexpression exacerbates apoptosis induced by myocardial ischemia-reperfusion injury in mice.

[0191] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The use of a reagent that inhibits the expression of the CIP gene and / or CIP protein in the preparation of a drug for treating myocardial infarction, characterized in that, The CIP gene has a Gene ID of 681849; The reagent includes siRNA targeting the CIP gene; The nucleotide sequence of the target sequence of the siRNA is shown in SEQ ID NO.5; The siRNA is selected from at least one of siRNA1 and siRNA2; the nucleotide sequence of the sense strand of siRNA1 is shown in SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.7; the nucleotide sequence of the sense strand of siRNA2 is shown in SEQ ID NO.8, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

9.

2. The application according to claim 1, characterized in that, The drug inhibits myocardial ischemia-reperfusion injury.

3. The application according to claim 1, characterized in that, The drug reduces oxidative stress damage to myocardial cells.

4. The application according to claim 1, characterized in that, The drug reduces cardiomyocyte apoptosis.

5. The application according to claim 1, characterized in that, The siRNA is siRNA1.

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