CN117159580A - Application of CIP inhibitor in preparation of medicines for treating myocardial infarction - Google Patents

Abstract

The invention discloses application of a CIP inhibitor in preparing a medicament for treating myocardial infarction, wherein the application comprises application of an agent for inhibiting CIP gene and/or CIP protein expression in preparing a medicament for treating myocardial infarction, treating heart ischemia reperfusion injury, reducing oxidative stress injury myocardial cells and reducing myocardial cell apoptosis. The invention proves that inhibiting CIP protein can relieve myocardial injury generated in the myocardial infarction process, reduce oxidative stress and myocardial cell apoptosis, promote myocardial cell proliferation, has great significance for treating ischemia reperfusion injury caused after myocardial infarction, and provides a new technical choice for clinical treatment of myocardial infarction.

Description

Application of CIP inhibitor in preparation of medicines for treating myocardial infarction

Technical Field

The invention relates to the technical field of biological medicine, in particular to application of an inhibitor of CIP in preparation of a medicament for treating myocardial infarction.

Background

Myocardial infarction is also called myocardial infarction, which refers to ischemic necrosis of cardiac muscle, and is characterized in that on the basis of coronary artery lesions, blood flow of coronary artery is rapidly reduced or interrupted, so that corresponding cardiac muscle is subjected to severe and persistent acute ischemia, and finally the ischemic necrosis of cardiac muscle is caused.

Myocardial infarction has become a cardiovascular and cerebrovascular disease which seriously damages the health of patients clinically, patients suffering from myocardial infarction often visit with the causes of poststernal pain and the like, and a clinical treatment method for myocardial infarction is to timely perform vascular recanalization, namely percutaneous coronary intervention, so as to save ischemic myocardium. However, the present research shows that myocardial cells are damaged during reperfusion after ischemia, so that the development of medicaments for treating reperfusion injury after ischemia is also of great importance for treating myocardial infarction.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides application of a CIP inhibitor in preparing medicines for treating myocardial infarction.

The first object of the present invention is to provide the use of an agent that inhibits the expression of CIP genes and/or CIP proteins in the preparation of a medicament for the treatment of myocardial infarction.

A second object of the invention is to provide the use of an agent that inhibits CIP gene and/or CIP protein expression in the manufacture of a medicament for treating cardiac ischemia reperfusion injury.

A third object of the present invention is to provide the use of an agent that inhibits CIP gene and/or CIP protein expression in the preparation of a medicament for alleviating oxidative stress injury to cardiomyocytes.

A fourth object of the invention is to provide the use of an agent that inhibits CIP gene and/or CIP protein expression in the preparation of a medicament for reducing myocardial apoptosis.

It is a fifth object of the present invention to provide a medicament for the treatment of myocardial infarction and/or cardiac ischemia reperfusion injury.

In order to achieve the above object, the present invention is realized by the following means:

experiments of in vivo and in vitro, knocking down and over-expression prove that the CIP protein plays a completely opposite function with heart failure and dilated cardiomyopathy in myocardial infarction for the first time, particularly, in the myocardial ischemia reperfusion process, the CIP protein promotes myocardial damage, and the CIP is inhibited from playing a protective role in the ischemia reperfusion damage process after myocardial infarction treatment.

The invention claims the following:

use of an agent that inhibits CIP gene and/or CIP protein expression in the preparation of a medicament for the treatment of myocardial infarction.

Use of an agent that inhibits CIP gene and/or CIP protein expression in the preparation of a medicament for treating cardiac ischemia reperfusion injury.

Use of an agent that inhibits CIP gene and/or CIP protein expression in the manufacture of a medicament for alleviating oxidative stress injury to cardiomyocytes.

Use of an agent that inhibits CIP gene and/or CIP protein expression in the preparation of a medicament for reducing cardiomyocyte apoptosis.

Preferably, the agent inhibits the expression of CIP genes and/or CIP proteins in the myocardium.

Preferably, the agent comprises siRNA targeting CIP gene.

More preferably, the nucleotide sequence of the targeting sequence of the siRNA is shown as SEQ ID NO. 5.

Further preferably, the siRNA is selected from at least 1 of siRNA1 and siRNA 2; the nucleotide sequence of the sense strand of the siRNA1 is shown as SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 7; the nucleotide sequence of the sense strand of the siRNA2 is shown as SEQ ID NO.8, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 9.

Still more preferably, the siRNA is siRNA1, the nucleotide sequence of the sense strand is shown as SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 7.

A medicament for treating myocardial infarction and/or cardiac ischemia reperfusion injury comprising siRNA targeting a CIP gene.

Preferably, the nucleotide sequence of the targeting sequence of the siRNA is shown as SEQ ID NO. 5.

More preferably, the siRNA is selected from at least 1 of siRNA1 and siRNA 2; the nucleotide sequence of the sense strand of the siRNA1 is shown as SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 7; the nucleotide sequence of the sense strand of the siRNA2 is shown as SEQ ID NO.8, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 9.

Further preferably, the siRNA is siRNA1, the nucleotide sequence of the sense strand is shown as SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 7.

Compared with the prior art, the invention has the following beneficial effects:

the invention proves that inhibiting CIP protein can relieve myocardial injury generated in the myocardial infarction process, reduce oxidative stress and myocardial cell apoptosis, promote myocardial cell proliferation, has great significance for treating ischemia reperfusion injury caused after myocardial infarction, and provides a new technical choice for clinical treatment of myocardial infarction.

Drawings

FIG. 1 is a graph showing CIP expression in a model of cardiac ischemia reperfusion injury; in the A mouse infarct model, the relative expression of CIP mRNA in the non-infarct zone (NIZ), infarct proximal zone (BIZ) and Infarct Zone (IZ); b is the relative expression of CIP mRNA in ischemia reperfusion injury model (IR) and Sham surgery group (Sham).

FIG. 2 shows the expression of CIP during hypoxia reoxygenation of cardiac myocytes; a is the mRNA level expression condition of CIP at different time points (1 h, 3h, 6h, 12h and 24 h) of primary milk mouse myocardial cell hypoxia for 6h reoxygenation; b is the expression of protein levels of CIP at different time points (1 h, 3h, 6h, 12h and 24 h) of primary milk mouse myocardial cell hypoxia for 6h reoxygenation.

Fig. 3 shows the change of cell viability of cardiomyocytes in which CIP was knocked down by hydrogen peroxide stimulation, and the knocking down of CIP can reduce cardiomyocyte death caused by oxidative stress.

FIG. 4 shows the expression of apoptosis-related proteins in myocardial cells stimulated by hydrogen peroxide to knock down CIP; a is the protein expression of Bax, bcl2 and clear amplified 3; b is the expression condition of AKT noumenon protein and phosphorylation protein.

FIG. 5 is a map of a pDC-MCMV-MCS-CMV-EGFP vector.

FIG. 6 is the effect of hydrogen peroxide stimulation on myocardial cells overexpressing CIP; a is the effect identification result of the over-expressed CIP; b is the post-core after overexpression of CIPDecreased muscle cell viability; c is H ₂ O ₂ Under the damage condition, the expression condition of the pro-apoptosis protein Bax and the anti-apoptosis protein Bcl2 after the overexpression of CIP.

FIG. 7 is the effect of knocking out CIP on cardiac ischemia reperfusion injury; a is protein expression result identification of knocking out CIP; b is the expression condition of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl2 of heart tissue in the ischemia reperfusion injury condition of the heart of CIP-KO.

FIG. 8 is the effect of knockdown CIP on protein kinase B (AKT).

FIG. 9 is the apoptotic effect of knockdown CIP on cardiac ischemia reperfusion injury; a is TUNEL detection of myocardial tissue apoptosis positive rate in ischemia reperfusion injury area, and green fluorescence indicates TUNEL positive myocardial nucleus; blue fluorescence indicates total cardiomyocyte nuclei; b is the statistical result of TUNEL apoptosis detection.

FIG. 10 is an apoptotic effect of over-expressed CIP on cardiac ischemia reperfusion injury, green fluorescence indicating TUNEL positive cardiomyocyte nuclei; blue fluorescence indicates total cardiomyocyte nuclei and red fluorescence indicates TNNT positive cardiomyocytes.

Detailed Description

The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

EXAMPLE 1 Down-expression of CIP in myocardial ischemia reperfusion injury model

1. Experimental method

1. Construction of cardiac ischemia reperfusion injury (I/R) model

Male rats (C57/BL 6) of 8-10 weeks of age were randomly divided into two groups, respectively designated as an operative group (i.e., IR group) and a sham operative group (i.e., sham group), wherein the operative group was a heart ischemia reperfusion injury (I/R) model, and the sham operative group was used as a control in accordance with the pathological change process of human myocardial infarction.

Surgical group: first, 1.5% sodium pentobarbital is used for intraperitoneal injection anesthesia, supine fixation, skin incision in the middle of the neck, and intubation, so that the rats can maintain normal breathing. Opening the chest cavity between the most obvious intercostals of heart pulsation, opening the ribs by using a face opener, tearing open the pericardium, pressing down the eyelid opener to enable the heart to pop out of the chest cavity, slightly holding the heart by using the thumb, the index finger and the middle finger of the left hand, taking the main trunk of the left coronary vein on the surface of the heart as a mark below the left auricle, penetrating the myocardial surface layer at the position 2mm below the Zuo Xin auricle by using a 5-0 noninvasive suture needle, and discharging the needle beside the pulmonary artery cone; after the heart recovers and stabilizes as a slipknot to ligate the anterior descending branch of the left coronary artery, the color of the corresponding area on the surface of the heart becomes dark to indicate that the ischemia is successful; after 45 minutes the ligature was loosened and the heart surface red indicating that the myocardial tissue restored to reperfusion. Gradually suturing rib, muscle and skin layer by layer, gradually reducing ventilation, removing trachea cannula after spontaneous breathing of the rat, observing whether secretion exists in the trachea, sucking secretion if so, suturing the trachea wound with 7-0 non-invasive suture needle, and gradually suturing neck muscle and skin layer by layer. The rats were placed from supine to right lateral position until they were awake.

Group of sham operations: substantially identical to the operation of the surgical suite, the only differences are: a5-0 noninvasive suture needle is used to pass through the surface layer of the cardiac muscle at the position 2mm below the root of the ear of Zuo Xin, and the needle is discharged beside the pulmonary artery cone to pass below the anterior descending branch of the left coronary artery without ligation.

2. Fluorescent quantitative PCR (polymerase chain reaction) detection of mRNA (messenger ribonucleic acid) expression condition of CIP (CIP)

(1) Drawing materials

The surgical group and the sham surgical group were obtained 24 hours after the end of the surgery, and the specific method was as follows:

after 8 weeks post-operative mice were subjected to cardiac ultrasound testing, they were anesthetized with 1-2% isoflurane. After anesthesia, the skin of the mice is cut along the lower sternum edge by making a transverse incision, and after the upper abdominal cavity is fully exposed, the diaphragm is cut. The heart was fully exposed by cutting the two ribs with scissors. The blood vessel at the bottom of the heart is fixed by forceps, the blood vessel at the bottom of the heart is cut off before the heart stops, the heart is rapidly placed in precooled PBS solution, and connective tissues around the heart and left and right atria are cut off when the heart pumps out residual blood in the heart cavity. After the ventricular tissue was weighed, it was immediately frozen in liquid nitrogen and transferred to-80℃for storage. After anterior descending blood vessel ligation, the area without blood supply (blushing) in the heart is an infarct area (IZ), the area within about 0.5cm around the infarct area (IZ) is a infarct adjacent area (BIZ), and other areas far away from the infarct area (IZ) and not affected by blood supply are non-infarct areas (NIZ).

(2) RNA extraction and reverse transcription

RNA from ventricular tissue was extracted according to conventional methods.

Solution system was configured (two 1.5mL EP tubes): system 1:5X gDNA Eraser buffer:2 μl x n (representing the number of specific EP tubes); gDNA Eraser:1 μl×n; and a second system: 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 x n.

Adding the calculated PCR water and RNA into the marked EP tube, and the total volume is 7 mu L; adding 3 mu L/hole system 1 into an EP tube added with water and RNA, vibrating, shaking uniformly, and lightly centrifuging; the PCR instrument was opened, the EP tube was placed in the middle, the PCR instrument lid was screwed down, and the procedure was set to: 42 ℃,2min, 10 mu L of reaction system; after the reaction is finished, taking out the EP tube, adding 10 mu L/hole reaction system 2, vibrating, shaking uniformly, and centrifuging; placing the sample into a PCR instrument, wherein the program is set as follows: 37 ℃ for 15min;85 ℃,5s;4℃and 20. Mu.L of the reaction system. Finally, the cDNA which is reverse transcribed is removed.

The resulting cDNA was used as a template for a fluorescent quantitative PCR reaction with primers shown in Table 1.

TABLE 1 fluorescent quantitative PCR primers

The fluorescent quantitative PCR reaction system is as follows: TB Green premix Ex Taq II,10 μl; cDNA, 0.2. Mu.L; water without ribozyme, 7.8 μl; ROX reference Dye,0.4 μl; upstream primer (10. Mu.M), 0.8. Mu.L; downstream primer (10. Mu.M), 0.8. Mu.L.

The fluorescent quantitative PCR reaction procedure was: 95 ℃ for 30s;95 ℃ for 5s; the plate was read at 60℃for 30s for 40 cycles. Dissolution profile analysis: the temperature is 55-95 ℃ and the reading is carried out once per minute.

2. Experimental results

As shown in a and B of fig. 1, the expression level of CIP protein was significantly decreased during ischemia reperfusion injury in rats of the surgical group, and the expression level of CIP was lower in infarct area than in non-infarct area, compared to sham surgical group. It is shown that in normal ischemia reperfusion injury, CIP protein plays a role in promoting injury, so that the body can correspondingly reduce CIP expression in order to relieve the injury.

EXAMPLE 2 downregulation of CIP expression during hypoxia reoxygenation of cardiomyocytes

1. Experimental method

1. Isolation and culture of primary Neonatal Rat Cardiomyocytes (NRCM)

(1) Separation of hearts

Alcohol spraying, breaking skin by using scissors, forceps and small scissors, and taking out heart of rat. The heart was placed in a petri dish with 1xPBS to remove excess tissue and blood stain, and transferred to a petri dish with 1xPBS on ice for temporary storage.

(2) Preparation of the solution

Preparing 4-tube pancreatin neutralization solution (namely DMEM (45 mL/tube) containing 10% (v/v) domestic serum (manufacturer: excel, product number: FSP 500) (5 mL/tube)), and preheating at 37 ℃ after preparation; preparing 2-tube primary digestive juice (namely solution A, the specific formula is NaCl,137mmol/L; KCl,4mmol/L; naHCO) ₃ 4.2mmol/L; glucose,5mmol/L; pH,7.68; filter sterilized) 30 mL/tube+0.25% trypsin (Trpsin) 15 mL/tube, 1 tube was prepared and preheated at 37 ℃.

3 tubes of cardiomyocyte culture fluid (namely DMEM 43.5 mL/tube, double antibody 0.5 mL/tube, transe serum 5 mL/tube, sodium pyruvate 0.5 mL/tube, glutamine 0.5 mL/tube) were prepared and mixed upside down.

(3) Tissue digestion

7mL of 1xPBS is added into a T50 conical flask, the index finger is wiped by sterilized paper, alcohol is sprayed into the table, the index finger is used as an operation table surface to hold the heart, the tissue is sheared into four parts by small scissors, and the four parts are placed into the conical flask. The flask was shaken several times with a tinfoil seal (containing the rotor) and the first non-tissue fluid was discarded in an ultra clean bench. 5mL of primary digestion solution preheated at 37 ℃ is added into a conical flask, and the tinfoil is sealed outside an ultra-clean bench for 5min by spiral oscillation at 37 ℃.

During digestion, a 50mL centrifuge tube was prepared, a 70 μm filter screen was fitted over the tube orifice, and pancreatin neutralization solution was added in an amount equivalent to that of the digestion solution. During the waiting period of digestion, gelatin (gelatin) can be used for plating, the addition amount is 1/2 of the volume of each cell hole, and the cells are incubated for at least 30min in a constant temperature incubator at 37 ℃.

When the first tube primary digest was used to the remaining 20mL, the second tube digest was prepared by preheating at 37 ℃. Whether the addition amount of the new primary digestive juice is increased or whether the digestion time is prolonged or not is determined according to the turbidity degree of the last digestive juice (generally, turbidity starts in the 3 rd to 5 th times of digestion), and the clearer the digestion strength is required to be enhanced until the digested tissue mass becomes a nasal discharge-like white connective tissue small particle, and repeated digestion can be stopped. In the digestion process, the times of digestion are recorded, the addition amount of the digestive juice and the digestion time are needed. The conical bottle needs to be held at the bottom of the bottle by part of fingers during transferring so as to prevent the bottle from falling vertically.

After tissue digestion, spraying alcohol into the table, discarding the first tissue digestion solution, and transferring the tissue digestion solution each time to a 70 mu m filter screen for filtration and neutralization. And adding new primary digestive juice into the conical flask, blowing off and uniformly mixing the agglomerated particles, and repeating the steps until all heart tissues are digested.

(4) Centrifuging

After digestion is complete, the filtered neutralized tissue digest is centrifuged at 1000rpm for 10min. During centrifugation, the plates (6-well plate, 1 mL/well; 12-well plate, 500. Mu.L/well; 24-well plate, 250. Mu.L/well) were incubated with gelatin, cell incubator 5% CO ₂ Incubate at 37℃for 0.5h.

(5) Wall-attached culture

The total volume of cardiomyocyte culture fluid that was finally required for homogenization, i.e. 5 hearts per 10cm dish, 10mL per 10cm dish, was calculated to determine the volume of heavy suspension for cell sedimentation. Removing supernatant in the centrifuge tube after centrifugation, adding myocardial cell culture solution for resuspension, and collecting cell sediment to 1 tube after multi-tube centrifugationEqually dividing into 10cm cell culture dishes, and placing into a cell culture box with 5% CO ₂ Incubating for 1-2 h at 37 ℃. This procedure is to pre-adhere fibroblasts to distinguish them from cardiomyocytes.

(6) Cardiomyocyte recovery

After the fibroblasts were attached to a 10cm cell culture dish, the supernatant was aspirated into a 50mL centrifuge tube, the dish was washed with fresh cardiomyocyte culture broth, and the broth was recovered to the centrifuge tube. After washing, the covered plate surface was observed with a microscope, and if there were a large number of residual cardiomyocytes (cardiomyocytes bright circles, fibroblast ash irregularities), washing was performed several times. And combining the obtained supernatant with a culture solution to obtain the NRCM suspension.

(7) Cardiomyocyte culture

Taking out the culture plate incubated with gelatin, discarding gelatin, homogenizing NRCM suspension, uniformly inoculating into cell culture plate at a six-well plate density of 1.5 newborn rats, culturing with complete medium (high sugar DMEM containing 10% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v) diantigen (P/S)) at 37deg.C and 5% CO ₂ Is cultured for 16 hours under the condition of (2).

2. Hypoxia reoxygenation experiment

(1) Preparation of anoxic buffer

The formulation of the myocardial anoxia buffer is: HEPES buffer 4mM,NaCl 117mM,KCl 12mM,CaCl ₂ 0.9mM，MgCl ₂ 0.49mM, sodium lactate 20mM, and buffer solution prepared by the method of reagent preparation, and mixed gas (containing 5% CO) ₂ And 95% N ₂ ) Slowly blowing through the buffer solution for 10min to remove dissolved oxygen in the buffer solution, thus obtaining the anoxic buffer solution.

(2) Anaerobic culture

NRCM was removed from the original medium, washed with PBS and replaced with hypoxia buffer. The hypoxic cells were placed in an anoxic incubator (i.e., nitrogen plus less than 1% oxygen) and incubated at 37℃for 6 hours, designated as anoxic group (i.e., hypoxia).

To 5% CO at 37 ℃C ₂ NRCM incubated for 6 hours under the conditions of (2) was used as a control and was designated normoxic group (i.e., normoxia).

(3) Reoxygenation culture

The anoxic group was removed from the anoxic incubator, the anoxic buffer was removed, and the anoxic group was replaced with a high sugar DMEM medium containing 10% (v/v) FBS, and the mixture was subjected to 5% CO at 37 ℃C ₂ 1h, 3h, 6h, 12h and 24h (i.e., R1h, R3h, R6h, R12h and R24 h) under the conditions of the culture medium to cause reoxygenation injury.

3. Fluorescent quantitative PCR (polymerase chain reaction) detection of mRNA (messenger ribonucleic acid) expression condition of CIP (CIP)

Total cellular RNAs of normoxic groups and anoxic groups of R1h, R3h, R6h, R12h and R24h were extracted and reverse transcribed to obtain cDNAs, respectively, according to the method of example 1.

The fluorescent quantitative PCR reaction was performed using the cDNA obtained by the reverse transcription as a template, and the primers used are shown in Table 1, and the fluorescent quantitative PCR reaction system and the fluorescent quantitative PCR reaction procedure were the same as in example 1.

4. Western blot detection of protein expression of CIP

The total cell proteins of normoxic groups and anoxic groups of R1h, R3h, R6h, R12h and R24h are respectively extracted, the protein concentration and protein denaturation are measured, western blot detection is carried out, and the information of the used antibodies is shown in Table 2.

TABLE 2 Western blot detection of antibodies for CIP

2. Experimental results

As shown in a in fig. 2, the CIP mRNA expression amount was reduced in the whole after reoxygenation in the hypoxic group compared to the normoxic group; as shown in B in fig. 2, the overall CIP protein expression was also reduced after reoxygenation in the hypoxic group compared to the normoxic group. It can be seen that both the RNA and protein of CIP are reduced under hypoxic-reoxygenation injury.

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

Example 3 knockdown of CIP to reduce myocardial apoptosis

1. Experimental method

1. Design of siRNA

Taking 1bp to 500bp of CDS sequence of CIP Gene (Gene ID: 681849) as target sequence (SEQ ID NO. 5), designing and obtaining 2 CIP specific siRNA (i.e. si-CIP-1-2), wherein the specific sequence is as follows:

si-CIP-1-sense strand: 5'-GGACCAGAGGAUACUGGAUTT-3' (SEQ ID NO. 6);

si-CIP-1-antisense strand: 5'-AUCCAGUAUCCUCUGGUCCTT-3' (SEQ ID NO. 7);

si-CIP-2-sense strand: 5'-GGCCCAAGUCUCUGGCUAUTT-3' (SEQ ID NO. 8);

si-CIP-2-antisense strand: 5'-AUAGCCAGAGACUUGGGCCTT-3' (SEQ ID NO. 9).

* Description of SEQ ID NOS.6 to 9 in the sequence Listing of the specification: according to the editing rules of WIPOSEQUEST software, the nucleotide sequence must only contain the symbols listed in the "WIPOST.26 annex I part 1", and the base "T" is "U" in the RNA sequence, so SEQ ID NO. 6-9 in the specification of the present invention are substantially identical to SEQ ID NO. 6-9 in the sequence table.

As a control, a general negative siRNA (i.e., si-NC, manufacturer: ji Ma gene, cat# A06001) was used.

2. Cell culture

H9C2 cardiomyocytes (Wohaze Life technologies Co., ltd.) were used at 1.0X10/ml ⁵ Individual cell densities were inoculated into six well plates, cultured in complete medium (high sugar DMEM containing 10% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v) double antibody (P/S)) at 37 ℃, 5% co ₂ Is cultured for 16 hours under the condition of (2).

3. Transfection

The growth of the cells was examined under a microscope, when the H9C2 cardiomyocytes were confluent at 60% -70%, the old medium was discarded, washed twice with PBS corresponding to the size of the dish, the corresponding amount of fresh medium (serum-free double antibody free) was placed in the dish, and siCIP-1-2 and siNC were transfected into H9C2 cardiomyocytes using RNAiMAX transfection reagent (manufacturer Invitrogen, cat no 13778), respectively, and the resulting cells were designated siCIP-1, siCIP-2 and siNC groups in this order. After 6 hours of transfection, the old medium was changed to new complete medium.

4. Hydrogen peroxide (H) ₂ O ₂ ) Stimulation(s)

48 hours after transfection, H was removed ₂ O ₂ Respectively adding into si-CIP-1 group, si-CIP-2 group and si-NC group at working concentration of 290 μm to obtain hydrogen peroxide stimulated group (H) ₂ O ₂ A group); serum-free high-sugar DMEM culture medium is used as a control and added into the si-CIP-1 group, the si-CIP-2 group and the si-NC group respectively, so that a control group (namely a Ctrl group) is obtained. After that, the culture was continued for 12 hours.

5. Cell viability assay (CCK 8 cytotoxicity experiment)

H ₂ O ₂ After the treatment is finished, the H9C2 myocardial cells are cleaned by serum-free DMEM, redundant hydrogen peroxide is removed, and the detection effect of CCK8 is prevented from being interfered. mu.L (96 well) of CCK-8 solution was added to each well, incubation was continued for 2 hours in a cell incubator, and absorbance was measured at 450 nm.

6. Western blot detection of expression of apoptosis-related proteins

(1) Description of apoptosis-related indicators

B lymphomas-2 gene (Bcl 2), BCL2-Associated X protein (Bax, bcl-2 family proteins directly regulate PTP). Among these proteins, the anti-apoptotic protein Bcl-2 is able to preserve the membrane potential, blocking the release of cytochrome C. And Bax is a pro-apoptotic protein that promotes the release of cytochrome C by affecting PTP to eliminate mitochondrial membrane potential. Therefore, the apoptosis degree of the cells can be measured by Bcl-2/Bax

Cysteine-aspartic protease (Caspase-3): caspases can directly disrupt cellular structures, such as cleavage of nuclear fiber layers. When apoptosis occurs in cells, laminin acts as a substrate and is cleaved by caspases at a near-middle fixed site, which disintegrates laminin and leads to the contraction of cellular chromatin. In addition, caspases inactivate or down-regulate enzymes, mRNA sheathes and DNA cross-linked proteins involved in DNA repair. Due to the action of DNA, these protein functions are inhibited, blocking proliferation and replication of cells and apoptosis. Caspase-3 is one of the Caspase proteases, and its increased protein expression greatly suggests increased apoptosis.

(2) Detection method

The total cell proteins of the si-CIP-1 group (i.e., siCIP1+H2O2) and the si-NC group (i.e., siNC+H2O2) of the hydrogen peroxide-stimulated group and the si-CIP-1 group (i.e., siCIP1+Ctrl) and the si-NC group (i.e., siNC+Ctrl) of the control group were extracted from the cell culture dish, western blot detection was performed as in example 2, and the information of the antibodies used is shown in Table 3.

TABLE 3 antibodies for Western blot detection of apoptosis-related proteins

2. Experimental results

As shown in fig. 3, the knockdown of CIP reduced cardiomyocyte death due to oxidative stress. Compared with a control group, the density of cells is increased after the stimulation of the hydrogen peroxide in the cells with the knockdown CIP, and the knockdown CIP relieves the H9C2 myocardial cell death induced by the hydrogen peroxide.

As shown in a in fig. 4, expression of pro-apoptotic protein Bax in H9C2 cardiomyocytes knocked down CIP was reduced and expression of apoptosis-inhibiting protein BcL-2 was increased, caspase-3 cutter expression was also increased, compared to control group under hydrogen peroxide stimulation; as shown in B in fig. 4, CIP knockdown group phosphorylated AKT increases in proportion under hydrogen peroxide stimulation.

The above results indicate that. Knocking down CIP reduces apoptosis caused by oxidative stress.

Example 4 overexpression of CIP to promote myocardial apoptosis

1. Experimental method

1. Construction of overexpression CIP vectors

Using mouse cardiac cDNA as template, using upstream amplification primers: 5'-ACCTCTCTCTCATTCTTTCACC-3' (SEQ ID NO. 10) and downstream amplification primers: 5'-CACTTCCACTCCAGCTTCC-3' (SEQ ID NO. 11), and amplified to obtain a CIP gene fragment (SEQ ID NO. 12) for constructing an over-expression vector.

The CIP gene fragment (SEQ ID NO. 12) was inserted between the BamHI site and the NotI site of the pDC-MCMV-MCS-CMV-EGFP vector (identical to "the adenoviral empty vector harboring the murine cytomegalovirus (mcV) promoter" in the PubMed document (PMID: 33677093)) shown in FIG. 5, to thereby obtain a CIP-overexpressing vector and a GFP-overexpressing vector, which were designated as an ad-CIP vector and an ad-GFP vector.

2. Adenovirus package

The ad-CIP vector and the pDC-MCMV-MCS-CMV-EGFP vector were submitted to Ji Kai gene company for adenovirus packaging, and CIP-overexpressing adenovirus (designated V-CIP) and control virus (designated V-GFP) were obtained in sequence.

3. Cell culture

H9C2 cardiomyocytes were cultured as in example 2.

4. Transfection

V-CIP and V-GFP were transfected into H9C2 cardiomyocytes, respectively, as in example 2, and the resulting cells were designated as ad-CIP and ad-GFP groups, respectively.

5. Western blot detection of protein expression of CIP

After 48h of transfection, western blot was used to detect CIP expression in the ad-CIP and ad-GFP groups as described in example 2.

6. Hydrogen peroxide (H) ₂ O ₂ ) Stimulation(s)

48H after transfection H ₂ O ₂ Adding into the ad-GFP group and the ad-CIP group respectively at working concentration of 290 μm to obtain hydrogen peroxide stimulated group (H) ₂ O ₂ A group); the high sugar serum-free DMEM medium was used as a control and added to the ad-GFP and ad-CIP groups, respectively, to obtain a control group (i.e., ctrl group). After that, the culture was continued for 12 hours.

7. Cell viability assay (CCK 8 cytotoxicity experiment)

For H, according to the procedure in example 2 ₂ O ₂ Group and ctrl group perform CCK8 detection.

8. Western blot detection of expression of apoptosis-related proteins

The total proteins of the cells of the ad-CIP group (i.e., ad-CIP+H2O 2) and the ad-GFP group (i.e., ad-GFP+H2O 2) of the hydrogen peroxide stimulated group and the ad-CIP group (i.e., ad-CIP+Ctrl) and the ad-GFP group (i.e., ad-GFP+Ctrl) of the control group were extracted. Western blot detection was performed as described in example 2, and the information on the antibodies used is shown in Table 3 in example 3.

2. Experimental results

As shown in a in fig. 6, CIP overexpression was successful. As shown in B in fig. 6, cardiomyocyte viability was decreased after CIP overexpression compared to the control group; as shown in C in fig. 6, the expression of CIP was overexpressed in the case of H2O2 injury, pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 compared to the control group.

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

Example 5 knock down of CIP to reduce apoptosis due to cardiac ischemia reperfusion injury

1. Experimental method

1. Construction of cardiac ischemia reperfusion injury (I/R) model

The CIP knockout mice (namely, "CIP-KO mice" in pubMed literature (PMID: 26436652)) and WT control mice (weight control at 23-28 g, raised in SPF-class animal culture room of the first Hospital affiliated with Zhongshan university) were randomly divided into two groups, and an operation group and a sham operation group were prepared respectively according to the method in example 1, namely, CIP knockout mice (KO+I/R) of the operation group, control mice (HET+I/R) of the operation group, CIP knockout mice (KO+sham) of the sham operation group, and control mice (HET+sham) of the sham operation group were obtained.

2. Western blot detection of expression of apoptosis-related proteins

The heart tissue was isolated 24 hours after the completion of the surgery from 4 groups of mice, protein samples were extracted, western blot was performed according to the method of example 2, and the information of the antibodies used was shown in Table 3 of example 3.

3. TUNEL apoptosis detection kit for detecting apoptosis

(1) Paraffin embedding

The heart tissue was isolated 24 hours after the completion of the surgery in 4 groups of mice, and paraffin tissue sections were prepared by fixing with paraformaldehyde and then delivering to the company (Severe Biotech Co.).

(2) Dewaxing

And placing the paraffin tissue slices on a metal slice baking frame, and placing the paraffin tissue slices in a baking oven at 65 ℃ for baking the slices for 1-2 hours, so that the slices are more firmly adhered and are not easy to strip, and dewaxing is facilitated.

In the pathology room, paraffin sections are sequentially soaked for 10min for the first time and 5min for the second time by passing through a glass jar containing xylene for 2 times, and the paraffin sections can be adjusted according to dewaxing conditions according to different seasons so as to be dewaxed fully. After dewaxing, hydrating, soaking the slices in 100% absolute ethanol for 5min, and repeating the steps once.

(3) Infiltration process

The slices are respectively soaked in ethanol with gradient (90%, 80% and 70%) for 1 time, and each time is 3-5 min. The hydrated slice is required to be rinsed for 2-3 times by using PBS, then the redundant moisture on the slide is removed by using a throwing piece mode, and if the moisture can not be removed, the water absorbing paper is used for carefully wiping to remove the redundant moisture around the sample, so that the sample can not be touched. To facilitate the liquid incubation process of the later steps, a hydrophobic, organized pen is used to draw a closed circle along the outline of the tissue. To avoid drying the samples during the experiment, the samples should be completed one by one and the end-of-the-loop samples placed in a wet box containing water.

(4) Penetrating through

And diluting the proteinase K solution with the concentration of 20 mu g/mL by using a PBS solution to obtain proteinase K diluent. The heart samples were thoroughly infiltrated with proteinase K dilutions and permeabilized in a wet box at room temperature for 20min.

The PBS solution washes the sample for 2-3 times, gently removes excess liquid, and carefully and gently wipes the liquid near the sample on the slide with absorbent paper to avoid touching the heart sample. Placing the samples with the ambient moisture absorbed in a wet box prevents the heart samples from being too dry.

(5) Labeling and TUNEL detection

The dried heart samples were tested according to the instructions using TUNEL apoptosis test kit (next holy biotechnology) as follows:

appropriate amount of 5X Equilibration Buffer was diluted with deionized water to give 1X Equilibration Buffer, and heart tissue was thoroughly soaked with 1X Equilibration Buffer at room temperature for 20min. Most of 1x Equilibration Buffer was washed off with absorbent paper, keeping the heart tissue moist, and moist paper towels or cotton could be used at the bottom of the box to avoid sloshing of the liquid.

Alexa Fluor 488-12-dUTP Labeling Mix stored at-20deg.C was dissolved on ice, enough TdT solution was prepared according to the instructions according to the number of samples, tdT incubation buffer was added dropwise to the heart samples, carefully placed into a black light-resistant wet box, transferred to an incubator and incubated at 37deg.C for 60min.

The cover glass was removed, and the tissue sections were placed in a 5 cm-position with PBS solution in the absence of light ² 50. Mu.L of TdT incubation buffer was added dropwise to the area of cells without allowing the cells to dry out. The slide should be protected from light during this later operation. To ensure uniform distribution of the reagents, a cover slip was used to gently cover the heart tissue. The shaker was slowly washed in a staining jar for 5min and again replaced clean PBS for 2 washes. PBS solution on the slice surface is removed by using a throwing piece mode, and residual water is gently erased by using water absorbing paper.

And dripping the prepared DAPI solution into the tissue sample area of the glass slide under the dark condition, and incubating for 6min at room temperature. After the cell nucleus is stained, the tissue sample is placed in a staining jar containing deionized water again for washing for 5min, and the tissue sample is washed again for 2 times after the liquid is replaced.

And (3) spin-drying excessive water on the glass slide, dripping anti-quenching sealing liquid around a sample area, covering a cover slip, sealing the periphery of the cover slip by using nail polish, and storing in a light-proof glass slide box.

The samples were observed using fluorescence microscopy at 520.+ -.20 nm and recorded by photographing.

2. Experimental results

As shown in a in fig. 7, CIP protein was not substantially expressed in mice and CIP knockout was successful. As shown in B in fig. 7, in CIP knockout mice, the expression amounts of the cardiac tissue pro-apoptotic protein Bax and the anti-apoptotic protein Bcl2 in the ischemic region were reduced in the case of cardiac ischemia reperfusion injury.

As shown in fig. 8, the protein kinase B (AKT) phosphorylation level of CIP knockout mice was increased, indicating that the CIP knockout activated AKT phosphorylation, promoting cell proliferation.

As shown in a and B in fig. 9, CIP knockout mice significantly decreased the number of YUNEL positive cells in the case of cardiac ischemia reperfusion injury, indicating that the number of apoptotic cells was decreased after ischemia reperfusion injury surgery in CIP knockout mice.

The results show that CIP protein is unfavorable to organisms in ischemia reperfusion injury, and knocking out CIP reduces apoptosis caused by heart ischemia reperfusion injury and relieves myocardial infarction.

Example 6 overexpression of CIP to reduce apoptosis due to cardiac ischemia reperfusion injury

1. Experimental method

1. Construction of cardiac ischemia reperfusion injury (I/R) model

CIP-conditioned over-expression mice (i.e., CIP in pubMed literature (PMID: 26436652)) at 8-10 weeks of age ^KI mice ") (weight was controlled at 23-28 g, and the mice were fed to SPF-class animal culture chambers of a first hospital affiliated to the university of Zhongshan) and randomly divided into two groups, one group was subjected to induction of cre enzyme and CIP overexpression by tamoxifen at 8 weeks to obtain CIP overexpressed mice (i.e., cre+ group), and the other group was not subjected to tamoxifen treatment to obtain control mice (i.e., cre-group).

The cre+ and cre-groups were treated as in the surgical group of example 1 to obtain the cre+ and cre-groups for cardiac ischemia reperfusion injury.

2. TUNEL apoptosis detection kit for detecting apoptosis condition

24 hours after the end of the surgery, heart tissues of cre+ and cre-groups were isolated and TUNEL apoptosis was detected as in example 5.

Cardiomyocytes in heart tissue of cre+ and cre-groups were also labeled with antibodies to skeletal muscle troponin (TNNT) (manufacturer: proteontech, cat. No. 15513-1-AP).

2. Experimental results

As shown in fig. 10, by TUNEL staining, it was found that the proportion of apoptosis caused by cardiac ischemia reperfusion injury was increased in CIP overexpressing mice compared to control mice. It was shown that overexpression of CIP aggravates apoptosis caused by ischemia reperfusion injury in the mouse heart.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims (10)

1. Use of an agent that inhibits CIP gene and/or CIP protein expression in the preparation of a medicament for the treatment of myocardial infarction.

2. Use of an agent that inhibits CIP gene and/or CIP protein expression in the preparation of a medicament for treating cardiac ischemia reperfusion injury.

3. Use of an agent that inhibits CIP gene and/or CIP protein expression in the manufacture of a medicament for alleviating oxidative stress injury to cardiomyocytes.

4. Use of an agent that inhibits CIP gene and/or CIP protein expression in the preparation of a medicament for reducing cardiomyocyte apoptosis.

5. The use according to any one of claims 1 to 4, wherein the agent comprises siRNA targeting CIP gene.

6. The use according to claim 5, wherein the nucleotide sequence of the targeting sequence of the siRNA is shown in SEQ ID No. 5.

7. The use according to claim 6, wherein the siRNA is selected from at least 1 of siRNA1 and siRNA 2; the nucleotide sequence of the sense strand of the siRNA1 is shown as SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 7; the nucleotide sequence of the sense strand of the siRNA2 is shown as SEQ ID NO.8, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 9.

8. The use according to claim 7, wherein the siRNA is siRNA1.

9. A medicament for treating myocardial infarction and/or cardiac ischemia reperfusion injury, comprising an siRNA targeting a CIP gene.

10. The drug of claim 9, wherein the nucleotide sequence of the targeting sequence of the siRNA is shown in SEQ ID No. 5.