CN113509471B - Application of AKT2 inhibitor in preparation of medicine for treating myocardial injury induced by doxorubicin - Google Patents

Abstract

The invention discloses the application of an AKT2 inhibitor in the preparation of a drug for treating myocardial injury caused by doxorubicin, and belongs to the technical field of biomedicine. In the present invention, in the myocardial injury model induced by doxorubicin, adult mice are injected with doxorubicin intraperitoneally, and the changes in diet and body weight are continuously observed, and then doxorubicin is given after interfering with the primary cardiomyocytes by an AKT2 inhibitor, and the detection is performed. The expression levels of oxidative stress-related genes were used to conclude that AKT2 inhibitors can reduce the cardiotoxicity caused by doxorubicin by enhancing the anti-oxidative stress ability of the heart.

Description

Application of AKT2 inhibitor in preparation of medicine for treating myocardial injury induced by doxorubicin

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to the application of an AKT2 inhibitor in the preparation of medicines for treating dilated cardiomyopathy and congestive heart failure caused by doxorubicin.

Background technique

About 9.6 million people worldwide die from cancer, and cancer is reported as the second leading cause of death worldwide. More than 600,000 women have died from breast cancer, accounting for about 15% of all cancers in women, according to the latest WHO figures. Chemotherapy, in addition to surgery, is an important means of fighting this devastating cancer. Despite the therapeutic benefits of chemotherapy, cardiotoxicity remains a serious problem to be addressed. Chemotherapy-induced cardiotoxicity has been reported to account for 7.25% to 27% of cardiovascular mortality in cancer patients receiving combination therapy. This evidence suggests that understanding how chemotherapy induces cardiotoxicity is critical for developing strategies to prevent related adverse effects. Doxorubicin (Dox) is one of the most common cardiotoxic chemotherapy known to cause breast cancer. Doxorubicin is a broad-spectrum anthracycline antineoplastic drug. Due to the strong affinity between doxorubicin and cardiolipin, it will gradually accumulate in the body while exerting antitumor effects. Causes cardiac dilation changes, and patients may develop left ventricular dysfunction, dilated cardiomyopathy, and heart failure after discontinuation of treatment.

AKT (protein kinase B (PKB)) is a serine/threonine protein kinase that is activated by many growth factors or cytokines in a phosphatidylinositol 3-kinase (PI3K)-dependent manner and mediates growth, including growth Various cellular responses, such as increased cell size, proliferation, survival, and glucose metabolism, among others. There are three subtypes of AKT in the mammalian genome, namely AKT1/PKBα, AKT2/PKBβ and AKT3/PKBγ, which are widely distributed but differentially expressed in various tissues. Under growth factor stimulation, AKT is phosphorylated at two of its regulatory phosphorylation sites, T308/S473 in AKT1, T309/S474 in AKT2 and T305/S472 in AKT3. Among all subtypes, AKT1 and AKT2 were expressed at the highest levels in the heart. Whereas AKT1 KO mice die embryonic due to severe developmental defects, AKT2 KO mice are able to grow to adults but exhibit mild growth defects. AKT2 KO mice exhibited marked hyperglycemia and insulin resistance, and developed type 2 diabetes, suggesting that AKT2 plays an important role in glucose-maintained homeostasis. Previous studies have shown that AKT2 can regulate the size of cardiomyocytes and promote the development of cardiomyocytes through the EndoG-MEF2A signaling pathway. Knockout of AKT2 also alleviates hypertensive heart disease induced by angiotensin 2 and high-salt diet. A large amount of data has confirmed that AKT2 plays an important role in myocardial cell injury, which provides a possibility for the study of the mechanism of the combined use of AKT2 and anti-tumor drug doxorubicin to improve the toxicity of doxorubicin.

Contents of the invention

The purpose of the present invention is to provide the application of AKT2 inhibitors in the preparation of drugs for the treatment of myocardial injury caused by doxorubicin, and to further explore whether the combination of AKT2 inhibitors and anticancer drug doxorubicin can significantly alleviate the toxic effect of cardiomyocytes, Lay the foundation for new drug development and targeted therapy.

It is well known to those skilled in the art that doxorubicin is a broad-spectrum anthracycline antineoplastic drug. Doxorubicin will gradually accumulate in the body while exerting its anti-tumor effect. Due to the strong affinity between doxorubicin and cardiolipin, doxorubicin can cause dilated cardiomyopathy and congestive cardiomyopathy when it accumulates to a certain dose in the body. Exhausted. The damage to the heart caused by doxorubicin is irreversible. In order to solve the problem of the toxicity of doxorubicin to the heart, the invention provides the application of an AKT2 inhibitor in the preparation of drugs for treating dilated cardiomyopathy and congestive heart failure caused by doxorubicin. The AKT2 inhibitor used in the present invention is CCT128930, which is an effective, ATP competitive and selective AKT2 inhibitor. The drug targets the AKT2 target 28-fold more selectively than its closely related PKA kinase.

Description of drawings

In Figure 1, A and C are the mitochondrial membrane potential of cardiomyocytes after administration of doxorubicin and AKT2 inhibitor CCT128930; B and D are the reactive oxygen species levels of cardiomyocytes after administration of doxorubicin and AKT2 inhibitor CCT128930; E is Primary cardiomyocytes stained with JC-1.

In Figure 2, A is DHE staining of primary cardiomyocytes; B is JC-1 staining of primary cardiomyocytes; C is the protein level of cardiac SOD in AKT2 KO mice after administration of doxorubicin; D is siAKT2 and primary cardiomyocytes Protein levels of AKT2 and SOD after doxorubicin treatment.

In Figure 3, A is 8-OH-dG staining of heart tissue sections; B is Sirius red staining of myocardial interstitium; C is Sirius red staining around myocardial vessels; D is SOD content of mouse heart; E is MDA of mouse heart content.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto. It should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

1. Materials and methods

1.1 Materials

Doxorubicin (C ₂₇ H ₂₉ NO _11` HCl, MW=579.98 g/mol) was added to 2 mL of MiliQ water to prepare a 5 mM Dox stock solution. Filter with a 0.22 μm filter head, aliquot and store in the dark at -20°C. Dilute 1:5000 with culture medium to make 1μM working solution.

The molecular weight of AKT2 inhibitor CCT128930 is 341.84, 5 mg powder is dissolved in 295 μL DMSO, the storage concentration is 50 mM mother solution, and it is diluted with medium 1:5000 to 10 μM working solution when used.

Rat-siAKT2 sequence: GGCAGGATGTGGTACAGAA (SEQ ID NO. 1).

PEI solution: Weigh 10mg of PEI, add 9ml of fresh cell-grade water, stir to dissolve. Adjust the pH value to 7.0, dilute to 10ml, filter with a 0.22μm filter, and store at 4°C after aliquoting.

10X electrophoresis: Weigh 144g Tris, 30.28g Gly, 10g SDS, and make up to 1L. When in use, add 450mL double-distilled water to every 50mL of 10X electrophoresis solution to prepare 1X electrophoresis solution for use.

10X electroporation: Weigh Tris 58g, Gly 29g, SDS 3.75g, and make up to 1L. When in use, add 100mL of methanol and 350mL of double distilled water to every 50mL of 10X electrotransfer solution to prepare 1X electrotransfer solution for use. After the 1X electro-transfer solution is prepared, put it in a -20°C refrigerator in advance to cool down.

10X TBS: Weigh 24.228g of Tris and 187.66g of NaCl, add 9mL of HCl, and dilute to 1L. When using, add 450mL of double-distilled water to every 50mL of 10X TBS, and then add 500μL of Tween 20 at a ratio of 1:1000, and mix thoroughly to obtain 1XTBST.

Protein lysate: Prepare 50mL protein lysate with 10mL SDS, 12.5mL 0.5M Tris-HCl and 27.5mL double distilled water.

5XLoading Buffer: Measure 0.3mL 1M Tris-HCl (pH=6.8), 1.25mL glycerol, 1mL 10% SDS and 5mg bromophenol blue, dilute to 5mL with double distilled water, aliquot into 1mL tubes and store at -20 ℃ in the refrigerator.

Naphthol Blue dye solution: measure 4mL of methanol, 0.8mL of acetic acid and 40mg of naphthol blue powder, and dilute to 40mL with double distilled water.

Naphthol Blue washing solution: measure 50mL of methanol and 7mL of acetic acid, and dilute to 100mL with double distilled water.

2. Method

2.1 Extraction and culture of primary cardiomyocytes from suckling rats

Primary cardiomyocytes were extracted from newborn 2-3 day old SD neonatal rats. The suckling rats were quickly decapitated, and the heart was taken out after thoracotomy, and quickly placed in the sterile 1XADS solution that had been cooled, and the remaining blood in the heart was squeezed out, and the atrium and heart were cut with ophthalmic scissors and ophthalmic forceps on the blood vessels. Use ophthalmic scissors to cut the ventricle into a plane, and try to cut it as much as possible but not shred it for easy digestion.

The following steps are performed in a biological safety cabinet. Collect every 5 hearts into 1.5mL EP tubes, add 950μL 1XADS and 12μL type II collagenase, and shake and digest at 12000rpm at 37°C for 15min. After the digestion is complete, transfer the digested cells to the same EP tube, and add an equal volume of medium to terminate the digestion. Centrifuge at 1000rpm for 5min, discard the supernatant and add 3mL medium to resuspend the cells. Repeat the above digestion steps three times, put the cell suspension collected four times into a centrifuge tube, transfer the cell suspension to a 100mm cell culture dish after the collection is completed, and carry out two differential attachment times, each attachment time is 45min. After the first differential attachment, the supernatant was transferred to a new 100mm dish, and after the second differential attachment, the supernatant was transferred to a new 15mL sterile centrifuge tube at 1000rpm for 5min. The centrifuged cells were resuspended with an appropriate amount of medium, and 10 μL of the cell suspension was stained with trypan blue for cell counting. The inoculated cell culture plate needs to be paved with 0.2% gelatin in advance, and discarded after standing for 30 minutes before cell inoculation. Before cell inoculation, mitotic inhibitors were added at a concentration of 50 ng/mL, and the medium was replaced with normal medium without mitotic inhibitors 5 hours later. Subsequent experiments can only be performed after the beating of primary cardiomyocytes is observed.

2.2 Transfection of primary cardiomyocytes with siAKT2

After the primary cardiomyocytes were cultured overnight and cell beating was observed, the prepared PEI solution was used to transfect siAKT2 (GGCAGGATGTGGTACAGAA). Before the start of the experiment, the PEI solution was taken out of the refrigerator to return to room temperature 1 hour in advance. Taking a 12-well plate as an example, add 2 μL PEI solution to a sterile centrifuge tube, then slowly add 5 μL siAKT2, gently blow and mix, and let stand at room temperature for 20 minutes. After 20 minutes, add 600 μL of serum-free medium with double antibody to the centrifuge tube, mix well and add to the cell culture plate. After 3 hours, an equal volume of medium with double serum was added and transfected for 24 hours.

2.3 Administration of primary cardiomyocytes

Doxorubicin and AKT2 inhibitor CCT128930 were diluted to the working concentration with the primary cell culture medium, the original medium in the cell culture plate was aspirated, and washed twice with PBS. The drug-added medium was added and cultured for 12 hours for subsequent detection.

2.4 Flow Cytometry

2.4.1 Detection of MitoSox by flow cytometry

Use MitoSox to detect the content of reactive oxygen species ROS in the mitochondria of cells. Each MitoSox powder was dissolved in 13 μL DMSO to make a 5 mM stock solution, which was diluted with PBS to a 2.5 μM working solution. The treated cells were digested with trypsin and transferred to a 1.5mL EP tube, centrifuged at 1500rpm for 5min, and the supernatant was discarded. Wash twice with PBS, centrifuge at 1500rpm for 5min each time. The pellet was resuspended with the diluted MitoSox working solution, and incubated at 37°C in the dark for 10 minutes. After the incubation, the cells were washed twice with PBS, and then the cells were resuspended in 500 μL PBS, passed through a 200-mesh sieve, and analyzed by flow cytometry.

After passing through the mesh, the cells were transferred to a flow tube and detected by a flow cytometer (BD FACSCelesta). Choose PE as the fluorescence channel. Set the gate to collect 20,000 cells and stop collecting. The first graph is plotted with FSC on the abscissa and SSC on the ordinate, and the gate is circled according to the distribution of cell populations in the graph. By adjusting the voltage values of the abscissa and ordinate, the cells are distributed in the lower left corner without pressing the line. Then draw an electropherogram, put the logical relationship of the electropherogram under the gate of the first circle, select PE on the abscissa, and select the number of cells on the ordinate. Adjust the voltage value of the FITC channel so that the output peak is at a suitable position. After all the settings are completed, the cells can be collected and the results can be analyzed with Flow Jo software.

2.4.2 Detection of DHE by flow cytometry

DHE was used to detect the content of reactive oxygen species Ros in the cells. DHE powder was dissolved in DMSO to make a 2mM stock solution, which was diluted with PBS to make a 5μM working solution. The cell treatment method was the same as that of MitoSox staining, and the incubation time was 30min. After passing through the mesh, the cells were transferred to a flow tube and detected by a flow cytometer (BD FACSCelesta). Choose PE as the fluorescence channel. Set the gate to collect 20,000 cells and stop collecting. The first graph is plotted with FSC on the abscissa and SSC on the ordinate, and the gate is circled according to the distribution of cell populations in the graph. By adjusting the voltage values of the abscissa and ordinate, the cells are distributed in the lower left corner without pressing the line. Then draw an electropherogram, put the logical relationship of the electropherogram under the gate of the first circle, select PE on the abscissa, and select the number of cells on the ordinate. Adjust the voltage value of the FITC channel so that the output peak is at a suitable position. After all the settings are completed, the cells can be collected and the results can be analyzed with Flow Jo software.

2.4.3 Detection of Rho123 by flow cytometry

When measuring mitochondrial membrane potential using Rho123. Rho123 powder was dissolved in DMSO to make 1mM stock solution, and diluted with PBS to make 1μM working solution. The cell treatment method was the same as that of MitoSox staining, and the incubation time was 20min. After passing through the mesh, the cells were transferred to a flow tube and detected by a flow cytometer (BD FACSCelesta). Fluorescent channel selects FITC. Set the gate to collect 20,000 cells and stop collecting. The first graph is plotted with FSC on the abscissa and SSC on the ordinate, and the gate is circled according to the distribution of cell populations in the graph. By adjusting the voltage values of the abscissa and ordinate, the cells are distributed in the lower left corner without pressing the line. Then draw an electropherogram, put the logical relationship of the electropherogram under the gate of the first circle, select FITC on the abscissa, and select the number of cells on the ordinate. Adjust the voltage value of the FITC channel so that the output peak is at a suitable position. After all the settings are completed, the cells can be collected and the results can be analyzed with Flow Jo software.

2.5 JC-1 staining

JC-1 is an ideal fluorescent probe widely used to detect mitochondrial membrane potential ΔΨm. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix (matrix), forming polymers (J-aggregates), which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondria In the substrate, JC-1 is a monomer at this time, which can produce green fluorescence. The relative ratio of red-green fluorescence is commonly used to measure the ratio of mitochondrial depolarization. The decline of mitochondrial membrane potential is a hallmark event in the early stage of apoptosis. The decrease of cell membrane potential can be easily detected by the transition of JC-1 from red fluorescence to green fluorescence, and the transition of JC-1 from red fluorescence to green fluorescence can also be used as an early detection index of apoptosis. The mitochondrial membrane potential of the cells was detected using Beyond's mitochondrial membrane potential kit (C2006).

Preparation of JC-1 staining working solution: take an appropriate amount of JC-1 (200X), and dilute JC-1 by adding 8 mL of ultrapure water to every 50 μL of JC-1 (200X). Vigorously vortex to fully dissolve and mix JC-1, then add 2mL JC-1 staining buffer (5X), and mix well to obtain JC-1 staining working solution.

Positive control setting: add CCCP (10mM) provided in the kit to the cell culture medium at a ratio of 1:1000, dilute to 10μM, and treat the cells for 20 minutes. For most cells, the mitochondrial membrane potential will be completely lost after 10 μM CCCP treatment for 20 minutes, and it should show green fluorescence after JC-1 staining; while normal cells should show red fluorescence after JC-1 staining.

For adherent cells (take a six-well plate as an example): Aspirate off the culture medium, wash once with an appropriate volume of PBS, and add 1 mL of cell culture medium. Cell culture medium may contain serum and phenol red. Add 1mL JC-1 staining working solution and mix thoroughly. Incubate at 37°C for 20 minutes in a cell culture incubator. During incubation, prepare an appropriate amount of JC-1 staining buffer (1X) by adding 4 mL of distilled water per 1 mL of JC-1 staining buffer (5X), and place in an ice bath. After the incubation, the supernatant was aspirated and washed twice with JC-1 staining buffer (1X) (according to the ratio of cell culture medium: JC-1 staining buffer (1X) = 1:2). Add 2 mL of cell culture medium (the culture medium may contain serum and phenol red). Since JC-1 stains living cells, the cells should be observed and photographed under a microscope immediately.

2.6 DHE staining

The ROS fluorescent probe DHE (Dihydroethidium, dihydroethidium) labels living cells and can detect the level of superoxide anion in the cells. DHE can enter the cell through the living cell membrane, and is oxidized by active oxygen in the cell to produce ethidium oxide, which can be embedded in chromosomal DNA to produce red fluorescence. According to the amount of red fluorescence generated in living cells, the content of ROS in cells can be judged.

Take the M4 as an example. Aspirate the medium in the culture plate and wash twice with PBS. Add 300 μL DHE (5 μM DHE working solution prepared with PBS) to each well, and incubate at 37° C. in the dark for 40 minutes. After incubation, the DHE staining solution was aspirated and washed three times quickly with PBS. Fix with 4% paraformaldehyde for 10 min, absorb the paraformaldehyde, and wash twice with PBS. Dilute DAPI with PBS at a ratio of 1:1000, and add 300 μL DAPI staining working solution to each well. Nuclear staining was carried out at 37°C for 15 minutes in the dark. Aspirate the DAPI and wash quickly 3 times with PBS. After staining, add a drop of anti-fluorescence quenching mounting medium to each well, cover with a circular cover slip, observe with a fluorescence microscope or laser confocal microscope, or temporarily store in a 4°C refrigerator in the dark for subsequent observation.

2.7 Animal models

The mice in this experiment are C57BL/6J strain, clean grade, purchased from Institute of Model Animals, Nanjing University. Set up wild-type mice (WT), wild-type doxorubicin-administered mice (WT-Dox), AKT2 gene knockout mice (AKT2 KO), AKT2 gene knockout doxorubicin-administered mice (AKT2 KO- Dox) four groups. WT-Dox and AKT2 KO-Dox mice were injected intraperitoneally with 2.5 mg/kg of doxorubicin, once every other day, and injected 6 times, with a cumulative dose of 15 mg/kg to establish the experimental model. After the administration of doxorubicin was stopped, the mice were fed normally for 12 days, and the body weight and food intake of the mice were recorded every other day during the modeling period. After 12 days, echocardiography was performed, and the whole blood and heart tissue of the mice were taken for detection.

2.8 Western Blot

2.8.1 Protein extraction

Prepare the total number of protein lysates required according to the number of samples, and add 100X protease inhibitor, 100X phosphatase inhibitor I and 100X phosphatase inhibitor II at a ratio of 1:100. Cut 20 mg of each heart tissue and place it in 500 μL of cold protein lysate, place it on ice, shred it fully on ice and homogenate. To extract cell protein, discard the medium in the culture dish, wash twice with PBS, add the prepared protein lysate, and collect the protein into a 1.5mL EP tube with a cell scraper. Place the EP tube containing the homogenate on ice, and use a sonicator to perform sonication (4°C, 32% power, super 5s, stop for 5s, total 40s for each sample). Centrifuge at 12,000 rpm for 20 min at 4°C, transfer the centrifuged supernatant to a new 1.5mL EP tube, and store at -20°C for later use.

2.8.2 Protein quantification

Protein quantification was performed using Novizyme's BCA protein concentration assay kit.

2.8.3 Western Blot

(1) Sample preparation: each protein was diluted according to the ratio of sample: 5XLoading Buffer = 4:1, 40 μg protein per well, and the sample preparation was made up to 20 μL with 1X Loading Buffer.

(2) Preparation of separating gel and stacking gel: prepare SDS-PAGE gel according to the ratio in the manual, generally use 10% separating gel, 12% separating gel can be prepared when the molecular weight is small, and 8% separating gel can be prepared when the molecular weight is large. Prepare the lower layer of separating gel first, use a pipette gun to add between the two glass plates as soon as possible, stop the glue filling when the distance from the top of the glass plate is about 2cm, take 500 μL of isopropanol and move along the edge of the short glass plate to form a liquid Seal and let stand at room temperature for 30 minutes, until the separating gel is polymerized and cross-linked, discard the isopropanol, and prepare the upper stacking gel. Use a pipette to pour the concentrated gel into the upper layer of the separating gel, and quickly insert the 10-hole comb to avoid air bubbles during the whole process. The gel-making equipment is placed at room temperature for 30 minutes, and the concentrated gel is polymerized and cross-linked before being transferred to electrophoresis. In the tank, follow-up protein loading and electrophoresis.

(3) SDS-PAGE gel electrophoresis: set up the electrophoresis device. Prepare 1× electrophoresis buffer and pour it into the electrophoresis tank. After adding, the liquid level in the electrophoresis tank is higher than the outer liquid level. Use a micro-loader to load the protein sample, concentrate at a constant pressure of 60V, and confirm that the sample enters the separation gel according to the protein marker band, adjust the voltage to 100V, and perform constant voltage electrophoresis until the 1X blue LoadingBuffer reaches the bottom of the separation gel, and then stop the electrophoresis .

(4) Membrane transfer: prepare 1× electrotransfer buffer in advance, pre-cool at -20°C for at least 1 hour, take a PVDF membrane with a length of 8.5 cm and a width of 5 cm, activate it with methanol for 1 min, wash it twice with double distilled water, and transfer it to the membrane Remove the stacking gel before cutting. Make a membrane transfer clip (cathode-absorbent sponge-filter paper-SDS-PAGE gel-PVDF membrane-filter paper-absorbent sponge-anode), 100V constant voltage, ice bath, transfer time is 90min, but when the molecular weight of the protein to be tested is large The transfer time needs to be extended.

(5) Blocking: 2% BSA blocking solution was prepared with 1XTBST, the PVDF membrane was taken out, and put into the blocking solution for 1 h.

(6) Apply the primary antibody: use 1X TBST solution to dilute the primary antibody according to the instructions, take out the PVDF membrane, cut it according to the protein marker band, put it into the incubation box, and incubate overnight at 4°C on a shaker.

(7) Membrane washing: The primary antibody was recovered the next day, the PVDF membrane was taken out, and washed 4 times with 1X TBST solution on a shaker, 10 min each time.

(8) Secondary antibody application: use 1% BSA solution to dilute the secondary antibody according to the instructions, put the PVDF membrane in the incubation box, and incubate at room temperature for 1 hour on a shaker.

(9) Membrane washing: the secondary antibody was not recovered, the PVDF membrane was taken out, and washed 4 times with 1X TBST solution on a shaker, 10 min each time.

(10) Exposure: Prepare the luminescent liquid according to the requirements of the ECL luminescent liquid kit, in which liquid A and liquid B are mixed at a ratio of 1:1 and protected from light for later use. After taking out the membrane, use absorbent paper to absorb the residual 1X TBST as much as possible, put the membrane protein side up, place it in a multifunctional gel imager, and evenly drop the exposure solution for exposure and shooting.

(11) Membrane dyeing: After the exposure, place the PVDF membrane in the naphthol blue dye solution and place it on a shaker for 5-10 minutes.

(12) Membrane washing: decolorize with naphthol blue washing solution after staining, wash twice, 5-10min each time. After washing, place the PVDF membrane on a white background and take pictures for preservation.

2.9 Pathological detection of mouse heart slices

2.9.1 8-OH-dG staining

8-Hydroxy-2'deoxyguanosine (8-OH-dG) is a product formed after the oxidation of reactive oxygen species and damage to nuclear DNA or mitochondrial DNA. After the heart was taken out, it was fixed in 4% paraformaldehyde fixative, and then the tissue was sent to Wuhan Xavier Biological Company for paraffin embedding, sectioning and staining. The slices were scanned and analyzed with a pathological slide scanner (NanoZoomer2.0RS). The higher the 8-OH-dG content, the darker the slice color.

2.9.2 Sirius Scarlet Dyeing

Collagen fiber is the most widely distributed and most abundant fiber in connective tissue, and is widely distributed in various organs. Both Sirius Scarlet and its lining solution are strong acid dyes, which are easy to combine with the basic groups in collagen molecules, and the adsorption is firm. Polarized light microscope detection shows that collagen fibers have positive uniaxial birefringence properties. When combined with Sirius Red composite staining solution, the birefringence can be enhanced and the resolution can be improved, thereby distinguishing the two types of collagen fibers.

The paraffin sections of heart tissue were dewaxed, stained in periodate staining solution for 15 min, and washed twice with tap water and double distilled water. The slices were placed in Schaffer staining solution, stained in the dark for 30 minutes, and rinsed with running water for 5 minutes. The slices were dyed in hematoxylin solution for 1-3min, washed with double distilled water, hydrochloric acid aqueous solution differentiated for a few seconds, washed with double distilled water, ammonia water solution turned blue, and washed with double distilled water. Finally, the sections were dehydrated and mounted. Sections are scanned using a pathology slide scanner, images are acquired and analyzed.

2.10 Detection of malondialdehyde in mouse heart tissue

In organisms, free radicals act on lipids to undergo peroxidation reactions, and the final product of oxidation is malondialdehyde (MDA), which can cause cross-linking and polymerization of biological macromolecules such as proteins and nucleic acids, and is cytotoxic, and is often used to evaluate oxidative damage. Thiobarbituric acid (TBA) method is often used to determine the content of lipid peroxide end product MDA in tissues. MDA and TBA are heated under acidic conditions to form a pink complex. The substance has a maximum absorption at a wavelength of 532nm, and the content of MDA can be determined by spectrophotometry according to this principle.

Take 50 mg of heart tissue from each mouse and add it to 450 μL of pre-cooled physiological saline, cut it into pieces and homogenize it to obtain 10% heart homogenate. Centrifuge at 6000rpm for 10min at 4°C, and take the supernatant. Add the following reagents in order according to the table below, and the specific operation steps are as follows:

After mixing the tubes, keep them away from light and put them in a water bath at 96°C. Take them out at regular intervals to see if the liquid turns red. When the liquid in the tubes turns red, take them out, put the tubes in ice water, and cool them down quickly. Take 200 μL of the liquid in each tube and place it on a microplate plate, and use a microplate reader to detect the OD value at a wavelength of 532 nm.

MDA content in heart tissue (nmol/mgprot) = (sample tube OD value - blank tube OD value) ÷ (standard tube OD value - blank tube OD value) x standard concentration (40nmol/mL) ÷ test sample protein concentration ( mgprot/mL)

2.11 Activity detection of superoxide dismutase (SOD) in mouse heart tissue

SOD in the sample can specifically scavenge superoxide anion radical O ^2- , and O ^2- can oxidize hydroxylamine to generate nitrite, which is purple-red under the action of the chromogen, and has a maximum absorption at 550nm. The activity levels of T-SOD (total SOD), CuZn-SOD and Mn-SOD were detected by superoxide dismutase (SOD) typing test kit (Nanjing Jiancheng).

Weigh 10 mg of heart tissue and place it in 1 mL of pre-cooled saline, cut it into pieces and homogenize it to prepare a 1% heart tissue homogenate solution. Centrifuge at 3000rpm for 15min at 4°C, and take the supernatant. Take 200 μL homogenate sample and add 200 μL Reagent VII, vortex and mix thoroughly for 1 min, centrifuge at 3500 rpm for 15 min at 4°C, and take the supernatant for CuZn-SOD determination.

After the reaction, 200 μL of the supernatant of the reaction solution was taken and placed in a microplate plate, and the OD value was measured by a microplate reader at a wavelength of 550 nm. In addition, BCA protein was used to quantify the protein concentration of each sample. Calculate the activity (U/mgprot) of total SOD and CuZn-SOD according to the formula, and calculate the enzymatic activity of Mn-SOD by subtraction.

Total SOD activity (U/mgprot) = (control OD value - measured OD value) ÷ control OD value ÷ 50% × dilution factor of the reaction system ÷ test sample protein concentration

CuZn-SOD activity (U/mgprot) = (control OD value - measured OD value) ÷ control OD value ÷ 50% × dilution factor of the reaction system ÷ test sample protein concentration

3. Statistical methods

All data of the present invention were processed with GraphPad Prism 5 (GraphPad Prism, San Diego, CA, USA) software and expressed as mean ± SEM values. Multiple groups were analyzed using Two-way ANOVA, and differences between the two groups were compared by unpaired t-test. Values of P<0.05 were considered statistically significant. "*" means p<0.05; "**" means p<0.01; "***" means p<0.001; "ns" means no significance, no significant difference.

4. Experimental results and conclusions

As shown in Figure 1, A indicates that doxorubicin can induce an increase in the level of oxidative stress in the mitochondria of cardiomyocytes, B indicates that doxorubicin can induce oxidative stress damage in cardiomyocytes, and C indicates that the AKT2 inhibitor CCT128930 can reduce the level of oxidative stress induced by doxorubicin The oxidative stress level of cardiomyocyte mitochondria, D means that doxorubicin can induce a significant increase in the level of ROS in cardiomyocytes, and the AKT2 inhibitor CCT128930 can significantly reduce the level of ROS in cardiomyocytes induced by doxorubicin. E: after administration of doxorubicin, the red fluorescence of cardiomyocytes weakens, and the green fluorescence increases; and after adding AKT2 inhibitors, the red fluorescence increases, indicating that the mitochondrial membrane potential of the cells decreases after administration of doxorubicin, and the AKT2 inhibitor CCT128930 can significantly Slow down the level of decrease in mitochondrial membrane potential of cells.

As shown in Figure 2, A and B, AKT2 inhibition can reduce the level of oxidative stress in cardiomyocyte mitochondria induced by doxorubicin. Doxorubicin can induce a significant increase in cardiomyocyte ROS levels, and AKT2 inhibition can significantly reduce the level of doxorubicin-induced cardiomyocyte ROS. C and D demonstrate that the inhibition of AKT2 can significantly increase the content of superoxide dismutase in the heart after doxorubicin injury at the animal and cell levels, respectively.

As shown in Figure 3, A indicates that doxorubicin can increase the content of 8-OHdG in the heart, and the inhibition of AKT2 can weaken the oxidation of reactive oxygen species induced by doxorubicin. B and C show that after doxorubicin treatment, WT mice have more pronounced myocardial interstitial and perivascular fibrosis than AKT2 KO mice, suggesting that inhibition of AKT2 attenuates doxorubicin-induced myocardial fibrosis. D shows that the SOD content of AKT2 KO mice was significantly increased after doxorubicin treatment. E shows that the MDA content of AKT2 KO mice was significantly reduced after doxorubicin treatment. The results showed that in doxorubicin-induced myocardial injury, AKT2 gene deletion promoted the expression of antioxidant genes and attenuated myocardial oxidative stress injury.

AKT2 inhibitors attenuate doxorubicin-induced myocardial injury, including decreased cardiac function and myocardial fibrosis. In addition, studies at the cellular level have re-verified that AKT2 gene knockout can alleviate cardiomyocyte death caused by doxorubicin. In order to further explore the mechanism of AKT2 gene deletion attenuating doxorubicin-induced myocardial injury, the effect of doxorubicin on the expression of oxidative stress-related genes in cardiomyocytes was detected after siAKT2 was used to interfere with the expression of AKT2 in cardiomyocytes. The results showed that the deletion of AKT2 could up-regulate the expression of the antioxidant gene SOD after doxorubicin treatment.

The above results indicated that AKT2 played an important role in the process of doxorubicin-induced myocardial injury. That is, the AKT2 inhibitor can reduce the toxic effect of doxorubicin on cardiomyocytes, thereby alleviating the myocardial injury caused by doxorubicin.

sequence listing

<110> China Pharmaceutical University

<120> Application of AKT2 inhibitors in the preparation of drugs for the treatment of myocardial injury induced by doxorubicin

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<170> SIPOSequenceListing 1.0

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<213> Artificial Sequence

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ggcaggatgt ggtacagaa 19

Claims (2)

1. Application of an AKT2 inhibitor in the preparation of a drug for the treatment and/or prognosis of myocardial injury induced by doxorubicin, the AKT2 inhibitor being the AKT2 inhibitor CCT128930 or siRNA; The sequence of the siRNA is: GGCAGGATGTGGTACAGAA. 2. The application according to claim 1, characterized in that: the myocardial injury caused by adriamycin is dilated cardiomyopathy or congestive heart failure.

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