Application of MAPK6 inhibitor in preparation of medicine for treating doxorubicin cardiotoxicity

Abstract

The invention provides application of an MAPK6 inhibitor in preparation of a medicine for treating doxorubicin cardiotoxicity, and belongs to the technical field of biological medicine. It is proved for the first time that MAPK6 and myocardial cell apoptosis caused by doxorubicin toxicity have important correlation, expression of myocardial cell apoptosis genes can be remarkably affected, then the apoptosis process is regulated and controlled, and the application prospect in the aspect of developing drugs for treating doxorubicin cardiotoxicity is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of a MAPK6 inhibitor in the preparation of a drug for treating doxorubicin-induced cardiotoxicity. Background Art

[0002] Doxorubicin (DOX) is a first-line anthracycline anti-cancer drug in clinical practice and is one of the most commonly used and effective drugs for treating hematological malignancies, solid sarcomas, cancers, etc. However, during clinical application, DOX can cause severe cardiotoxicities such as a decrease in left ventricular ejection fraction (LVEF), tachycardia, arrhythmia, cardiomyopathy, and heart failure (grades III-IV). Epidemiological studies have shown that the incidence of cardiotoxicity in patients receiving DOX treatment can be as high as 18%. In addition, a study of childhood cancer survivors shows that more than 50,000 childhood cancer survivors will face the risk of cardiovascular diseases due to the use of DOX. This indicates that DOX-induced cardiotoxicity has seriously threatened human life and health.

[0003] DOX-induced cardiotoxicity involves pathological mechanisms such as oxidative stress, mitochondrial dysfunction, and apoptosis. Myocardial cell apoptosis is a highly regulated form of programmed cell death. Bax, Bcl-2, and caspases are important regulatory factors during the process of cell apoptosis, which are the main pathological mechanisms of DOX-induced cardiotoxicity, are closely related to the generation of reactive oxygen species (ROS), and are the key points for intervening in DOX-induced cardiotoxicity.

[0004] Although the pathology of DOX-induced cardiotoxicity has been well studied, the current research on the targets of DOX-induced cardiotoxicity still needs to be deepened, and there is also a lack of effective targets and therapeutic drugs for myocardial cell apoptosis. Therefore, studying the targets of DOX-induced cardiotoxicity and deeply revealing the biological basis and regulatory mechanisms of DOX-induced cardiotoxicity are of great significance for clarifying its pathogenic mechanism and developing effective intervention means.

[0005] Based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0006] MAPK6 (Mitogen-Activated Protein Kinase 6) is an atypical mitogen-activated protein kinase and belongs to the MAPK family. MAPK6 plays an important role in biological processes such as cell proliferation and differentiation. In addition, the activation of MAPK6 can respond to various external stimuli, such as growth factors, cytokines, and oxidative stress, etc. The present invention also first proves that there is an important association between MAPK6 and myocardial cell apoptosis, which can significantly affect the expression of myocardial cell apoptosis genes, and further regulate the apoptosis process, and has application prospects in the development of apoptosis drugs for myocardial cells.

[0007] Based on this, in order to provide more drugs for intervening in DOX cardiotoxicity, the present invention provides the following technical solutions:

[0008] The present invention provides the use of a MAPK6 inhibitor in the preparation of a drug for treating doxorubicin cardiotoxicity.

[0009] Preferably, the doxorubicin cardiotoxicity refers to cardiomyocyte apoptosis induced by doxorubicin.

[0010] Preferably, the MAPK6 inhibitor is an shRNA sequence that inhibits MAPK6 expression.

[0011] Preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO: 1.

[0012] SEQ ID NO: 1: ATCCTTACATGAGCATATATT.

[0013] The present invention also provides a drug for treating doxorubicin cardiotoxicity in the above application. The drug is a biological preparation containing an shRNA that inhibits MAPK6 expression, and the nucleotide sequence of the shRNA is as shown in SEQ ID NO: 1.

[0014] Preferably, the dosage form of the biological preparation includes injection preparations, powders, and oral liquid preparations.

[0015] The present invention has found through research that cardiomyocyte apoptosis is a key link in the intervention of DOX cardiotoxicity, and MAPK6 plays an important role in cardiomyocyte apoptosis and is a key target for intervening in cardiomyocyte apoptosis. At the same time, Shengmai injection, as a MAPK6 inhibitor, can inhibit apoptosis, has high safety, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a result graph showing an increase in the mRNA expression level of apoptotic factors in cardiomyocytes overexpressing MAPK6 (***: p < 0.001; **: p < 0.01; *: p < 0.05; N = 3); wherein:

[0017] Figure 1 A in is a result graph of the protein expression level of MAPK6 in cells;

[0018] Figure 1 B in is a result graph of cell viability;

[0019] Figure 1 C in is a result graph of the mRNA expression level of apoptotic factor BAX;

[0020] Figure 1 D in is a result graph of the mRNA expression level of apoptotic factor BCL2;

[0021] Figure 1 In which, E is the result graph of the mRNA expression level of apoptosis factor CASP3;

[0022] Figure 1 In which, F is the result graph of flow cytometry for cell apoptosis;

[0023] Figure 1 In which, G is the quantitative analysis result of the live cells in Figure 1 F in;

[0024] Figure 1 In which, H is the result graph of the intracellular ROS level;

[0025] Figure 1 In which, I is the quantitative analysis result of the ROS in Figure 1 H in;

[0026] Figure 1 In which, J is the result graph of the protein expression level of DRP1 in cells overexpressing MAPK6;

[0027] Figure 1 In which, K is the WB quantitative analysis result of DRP1 in Figure 1 J in;

[0028] Figure 2 Is the result graph of the decreased mRNA expression level of apoptosis factor in cardiomyocytes with MAPK6 knockdown (***: p < 0.001; **: p < 0.01; *: p < 0.05; N = 3); Among them:

[0029] Figure 2 In which, A is the result graph of the protein expression level of MAPK6 in cells;

[0030] Figure 2 In which, B is the result graph of cell viability;

[0031] Figure 2 In which, C is the result graph of the mRNA expression level of apoptosis factor BAX;

[0032] Figure 2 In which, D is the result graph of the mRNA expression level of apoptosis factor BCL2;

[0033] Figure 2 In which, E is the result graph of the mRNA expression level of apoptosis factor CASP3;

[0034] Figure 2 In which, F is the result graph of the decreased flow cytometry for cell apoptosis;

[0035] Figure 2 In which, G is the Figure 2 Quantitative analysis result of the live cells in F in;

[0036] Figure 2 In which, H is the result graph of the decrease in intracellular ROS level;

[0037] Figure 2 In which, I is the quantitative analysis result of the ROS in H in Figure 2 ;

[0038] Figure 3 is the result graph of detecting the interaction between DOX and MAPK6 by surface plasmon resonance technology; wherein:

[0039] Figure 3 In which, A is the SDS-PAGE result graph of purified MAPK6 protein;

[0040] Figure 3 In which, B is the result graph of detecting the interaction between DOX and MAPK6 by surface plasmon resonance technology.

[0041] Figure 4 Result graph of Shengmai injection inhibiting MAPK6 and then reducing apoptosis of cardiomyocytes (***: p < 0.001; **: p < 0.01; *: p < 0.05; N = 8), wherein:

[0042] Figure 4 In which, A is the result graph of detecting the expression level of MAPK6 protein in heart tissue inhibited by Shengmai injection by WB;

[0043] Figure 4 In which, B is the WB quantitative analysis result of MAPK6 in A in Figure 4 ;

[0044] Figure 4 In which, C is the result graph of detecting the expression level of MAPK6 mRNA in heart tissue inhibited by Shengmai injection by qPCR;

[0045] Figure 4 In which, D is the result graph of detecting the inhibition of cardiomyocyte apoptosis by Shengmai injection by Tunel;

[0046] Figure 4 In which, E is the quantitative analysis result graph of apoptotic cells in D in Figure 4 ;

[0047] Figure 4 In which, F is the result graph of detecting the expression level of BAX protein in heart tissue inhibited by Shengmai injection by WB;

[0048] Figure 4 In which, G is the WB quantitative analysis result graph of BAX in F in Figure 4 ;

[0049] Figure 4The "H" in it is the result graph of qPCR detecting the inhibitory effect of Shengmai Injection on the expression level of BAX mRNA in cardiac tissue;

[0050] Figure 5 It is the result graph of qPCR detecting the increase in the expression level of NRF2 mRNA in cardiac tissue by Shengmai Injection;

[0051] Figure 6 It is a representative image of M-mode echocardiogram of Shengmai Injection improving cardiac function;

[0052] Figure 7 It is the result graph of Shengmai Injection increasing EF%;

[0053] Figure 8 It is the result graph of Shengmai Injection increasing FS%; Detailed implementation mode

[0054] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1 Overexpression of MAPK6 affects the increase in DOX-induced cardiomyocyte apoptosis

[0056] The specific method is as follows:

[0057] (1) Construction of MAPK6 overexpression plasmid and lentivirus packaging

[0058] The overexpression plasmid of MAPK6 was synthesized by GenScript. The synthesized plasmid was transformed into stb13 competent cells, and the plasmid was extracted according to the instructions of the plasmid large extraction kit.

[0059] 293T cells cultured in a 10 cm dish were digested and inoculated into 6 T25 cell culture flasks at a ratio of 1:6. When the cell confluence reached 90%, they were used for virus packaging. Virus packaging system: Solution A: 0.9 mL of Opti-MEM, 28 μL of PEI; Solution B: 0.9 mL of Opti-MEM, 3.5 μL of psPAX2 plasmid, 3.5 μL of pMD2.G, 7 μL of the target plasmid. Solution A was slowly added to Solution B, gently mixed, and left standing at room temperature for 15 min.

[0060] After discarding 1.8 mL of the original culture medium in the T25 culture flask, evenly dropwise add the above-mentioned incubated AB mixture into the culture dish, gently mix it, and place it in the incubator for culture. After culturing for 6 h, change the culture medium, add 6 mL of fresh complete DMEM culture medium, continue culturing for 24 h, and then collect the first virus supernatant. Redropwise add 6 mL of complete cell culture medium of DMEM, continue culturing for 24 h, and collect the second virus supernatant. Mix the virus supernatants collected twice, centrifuge at 4°C and 500 g for 10 min, filter to remove impurities using a 0.45-μm filter, and temporarily store it in a 4°C refrigerator.

[0061] (2) Construct AC16 cells overexpressing MAPK6

[0062] Seed 1.0×10 5 AC16 cells in a 6-well plate, place it in an incubator at 37°C and 5% CO 2 After culturing for 24 h, change the old culture medium to fresh culture medium containing 8 μg / mL polybrene. For the group overexpressing MAPK6 (AC16-OE-MAPK6), add 1 mL each of fresh culture medium containing 8 μg / mL polybrene and virus; for the wild-type group (AC16-OE-Ctrl), add 2 mL of fresh culture medium containing 8 μg / mL polybrene. Discard the culture medium after infection for 24 h and change to fresh culture medium. After infection for 48 h, change the culture medium to complete DMEM / F-12 culture medium containing 1.0 μg / mL puro, screen for 14 days, collect the cells, and detect the expression level of the target protein by WB.

[0063] (3) Detect the expression level of MAPK6 in AC16 cells by WB

[0064] AC16 cells and AC16 cells overexpressing MAPK6 were washed with PBS, digested with 0.25% trypsin for 2 min, terminated with 2 mL of DMEM / F-12 complete medium, collected, washed thoroughly with PBS, and the residual liquid was blotted dry. 200 μL of RIPA lysis buffer (RIPA:PMSF:cocktail phosphatase inhibitor = 100:1:1) was added, and the cells were lysed on ice for 20 min, pipetted and mixed every 3 min, centrifuged (4 °C, 12,000 rpm, 15 min), and the supernatant was collected. The protein concentration of the cell lysate was detected by a BCA kit. 5× Loading buffer was added to the protein sample in proportion, mixed well, and denatured at 95 °C for 10 min. The loading amount per well was 40 μg, and the concentration of the separating gel was 10%. The electrophoresis conditions were: constant voltage of 100 V for 80 min. When the bromophenol blue band reached the bottom of the separating gel, the electrophoresis was terminated. A 0.45 μm PVDF membrane was pre-activated with methanol for 5 min. After electrophoresis, the stacking gel part of the gel was cut off and the front side was marked, and then the gel, PVDF membrane, and filter paper were transferred to the transfer buffer for equilibration. A sandwich (blackboard, filter paper, gel, PVDF membrane, filter paper) was prepared and placed in the transfer tank, and transferred at a constant current of 200 mA for 90 min. Blocked with 5% non-fat milk powder at room temperature for 60 min. Incubated with MAPK6 and GAPDH antibodies overnight on a shaker at 4 °C, washed 3 times with TPBS, incubated with rabbit anti at room temperature for 30 min, and developed after washing 3 times with TPBS.

[0065] (4) Detection of the viability of AC16 cells overexpressing MAPK6 by CCK8

[0066] 1.0×10 5 AC16-OE-Ctrl and AC16-OE-MAPK6 cells were seeded into 96-well plates respectively, and each type of cell was divided into 2 groups: Ctrl group and 0.5 μM DOX group, with 6 replicates in each group. They were cultured at 37 °C and 5% CO 2 for 24 h until the cell confluence reached 80%. The DOX group was given complete medium containing 0.5 μM DOX, and the control group was given complete medium with solvent control, and cultured for 48 h. The original culture medium was discarded, 100 μL of complete medium and 10 μL of CCK8 reaction reagent were added respectively, and reacted at 37 °C and 5% CO 2 for 60 min, and the OD450 value was measured with an enzyme-labeling instrument to calculate the cell viability.

[0067] (5) Detection of the expression level of apoptotic factor mRNA in AC16 cells overexpressing MAPK6 by qPCR

[0068] The AC16 cells overexpressing MAPK6 were counted, and 1×10 5Cells were seeded in 6-well plates. Each type of cell was divided into 2 groups: Ctrl group and 0.5 μM DOX group, with 3 replicate wells in each group. They were cultured at 37 °C and 5% CO 2 for 24 h until the cell confluence reached 80%. The total RNA of cells in different treatment groups was extracted using the Total RNA Isolation Kit V2 from Novoprotein. The RNA concentration was measured by Nanodrop 2000. 1 μg of RNA sample was added with 2 μL of 4×DNA remover Mix (containing gDNA remover), and the volume was made up to 8 μL with DEPC water. The reaction was carried out at 37 °C for 5 min. The reactant was added with 2 μL of 5×M5 RT Super Mix, incubated at 42 °C for 15 min, heated at 96 °C for 5 min, cooled on ice, and the RT product was diluted twice with DEPC water as the qPCR template. The qPCR reaction system was as follows: 95 °C for 10 s; 60 °C for 30 s, for 40 cycles; Dissociation curve: Heat-treated gradually from 60 °C to 95 °C at a rate of 0.05 °C / s, and 2 -ΔΔCt was used to calculate the gene expression levels of each group. The qPCR primer sequences are shown in Table 1.

[0069] Table 1 PCR primer sequences

[0070]

[0071]

[0072] Note: All species are human.

[0073] (6) Detection of apoptosis of AC16 cells overexpressing MAPK6 by flow cytometer

[0074] 2.5×10 4 AC16-OE-Ctrl and AC16-OE-MAPK6 cells were seeded in 24-well plates respectively. Each type of cell was divided into 2 groups: Ctrl group and 1.0 μM DOX group, with 3 replicate wells in each group. They were cultured at 37 °C and 5% CO 2Cultivate for 24 h under the conditions until the cell confluence reaches 80%. For DOX, add complete medium containing 1.0 μM DOX, and for the control group, add complete medium with solvent control, and cultivate for 36 h. Digest the cells, centrifuge to collect the cells, resuspend the cells with 100 μL of pre-cooled PBS and transfer them to the V-bottom plate for flow cytometry, centrifuge (4 °C, 2000 rpm, 4 min), discard the waste liquid, repeat washing the cells with 100 μL of pre-cooled PBS, discard the supernatant, and add 100 μL of Annexin V Binding Buffer (1×) to resuspend the cells. Add 2.5 μL of Annexin V-APC Reagent and 2.5 μL of 7-AAD Reagent (100 μg / mL) to the cell suspension. Gently pipette and mix the cells with a multi-channel pipette. Incubate in the dark at room temperature for 15 min. Add 300 μL of Annexin V Binding Buffer (1×) and mix the sample. Immediately detect on the machine.

[0075] (7) Detection of apoptosis of AC16 cells overexpressing MAPK6 by fluorescence microscopy

[0076] 1.0×10 4 AC16-OE-Ctrl and AC16-OE-MAPK6 cells were respectively seeded into 48-well plates, and each type of cell was divided into 2 groups: Ctrl group and 1.0 μM DOX group, with 3 replicates in each group. Cultivate at 37 °C, 5% CO 2 under the conditions for 24 h until the cell confluence reaches 80%. For DOX, add complete medium containing 1.0 μM DOX, and for the control group, add complete medium with solvent control, and cultivate for 36 h. Dilute the stock solution to a 5 μM working solution with serum-free medium before use (the ratio of staining solution to dilution solution is 1:1000). Add 200 μL of the 5 μM working solution to each well and cultivate at 37 °C, 5% CO2 for 30 min. Discard the staining solution, wash the cells 2 times with 1×PBS, and detect with a fluorescence microscope.

[0077] The results are as Figure 1 shown. The protein level of MAPK6 in AC16 cells overexpressing MAPK6 increased significantly (as shown in Figure 1 A), the cell viability decreased significantly (as shown in Figure 1 B, p < 0.05), and the expression levels of related apoptosis genes BAX (as shown in Figure 1 C, p < 0.001), BCL2 (as shown in Figure 1 D, p < 0.01), and CASP3 (as shown in Figure 1 E, p < 0.05) mRNA increased significantly. The apoptosis of AC16 cells overexpressing MAPK6 increased significantly (as shown in Figure 1 F and G). After overexpressing MAPK6, the intracellular ROS level decreased significantly (as shown inFigure 1 As shown in H and I, p < 0.05). In addition, after overexpressing MAPK6, the expression level of DRP1 in cells increased significantly (as Figure 1 shown in J and K, p < 0.05).

[0078] Example 2 Knockdown of MAPK6 Inhibits DOX-Induced Apoptosis of Cardiomyocytes

[0079] The specific method is as follows:

[0080] (1) Construction of MAPK6 Knockdown Plasmid and Lentivirus Packaging

[0081] The MAPK6 knockdown plasmid was synthesized by GenScript, and the shRNA is shown in SEQ ID NO: 1. The synthesized plasmid was transformed into stb13 competent cells, and the plasmid was extracted according to the instructions of the plasmid large extraction kit. Virus packaging was carried out according to the method in (1) of Example 1.

[0082] (2) Construction of AC16 Cells with Knockdown of MAPK6

[0083] 1.0×10 5 AC16 cells were seeded in 6-well plates and cultured at 37°C in a 5% CO 2 incubator for 24 h. Then, the old medium was replaced with fresh medium containing 8 μg / mL polybrene. For the MAPK6 knockdown group (AC16-KD-MAPK6), 1 mL of fresh medium containing 8 μg / mL polybrene and virus were added respectively. For the wild-type group (AC16-KD-Ctrl), 2 mL of fresh medium containing 8 μg / mL polybrene were added. After 24 h of infection, the medium was discarded and replaced with fresh medium. After 48 h of infection, the medium was changed to DMEM / F-12 complete medium containing 1.0 μg / mL puro, and screening was carried out for 14 days. The cells were collected, and the expression level of the target protein was detected by WB.

[0084] The experimental procedures of WB detection, CCK8 detection, qPCR detection, flow cytometry analysis, and ROS detection were the same as those in Example 1.

[0085] The results are as Figure 2 shown. The protein level of MAPK6 in AC16 cells with knockdown of MAPK6 decreased significantly (as Figure 2 shown in A), the cell viability increased significantly (as Figure 2 shown in B, p < 0.05), and the related apoptotic genes BAX (as Figure 2 shown in C, p < 0.001), BCL2 (as Figure 2 shown in D, p < 0.001), CASP3 (as Figure 2As shown in E, the expression level of p<0.001)mRNA increased significantly. The apoptosis of AC16 cells with MAPK6 knockdown decreased significantly (as Figure 2 shown in F and G, p<0.01). In addition, after MAPK6 knockdown, the intracellular ROS level decreased significantly (as Figure 2 shown in H and I, p<0.01).

[0086] Example 3 Detection of the interaction between DOX and MAPK6 by surface plasmon resonance technology

[0087] The specific method is as follows:

[0088] (1) Construction of MAPK6 expression vector: Start codon (M) + N-His-tag (HHHHHH) + target protein (SEQ ID NO: 10) + stop codon (*). The sequence is as follows: MHHHHHHAEKFESLMNIHGFDLGSRYMDLKPLGCGGNGLVFSAVDNDCDKRVAIKKIVLTDPQSVKHALREIKIIRRLDHDNIVKVFEILGPSGSQLTDDVGSLTELNSVYIVQEYMETDLANVLEQGPLLEEHARLFMYQLLRGLKYIHSANVLHRDLKPANLFINTEDLVLKIGDFGLARIMDPHYSHKGHLSEGLVTKWYRSPRLLLSPNNYTKAIDMWAAGCIFAEMLTGKTLFAGAHELEQMQLILESIPVVHEEDRQELLSVIPVYIRNDMTEPHKPLTQLLPGISREALDFLEQILTFSPMDRLTAEEALSHPYMSIYSFPMDEPISSHPFHIEDEVDDILLMDETHSHIYNWERYHDCQFSEHDWPVHNNFDIDEVQLDPRALSDVTDEEEVQVDPRKYLDGDREKYLEDPAFDTNYSTEPCWQYSDHHENKYCDLECSHTCNYKTRSSSYLDNLVWRESEVNHYYEPKLIIDLSNWKEQSKEKSDKKGKSKCERNGLVKAQIALEEASQQLAGKEREKNQGFDFDSFIAGTIQLSSQHEPTDVVDKLNDLNSSVSQLELKSLISKSVSQEKQEKGMANLAQLEALYQSSWDSQFVSGGEDCFFINQFCEVRKDEQVEKENTYTSYLDKFFSRKEDTEMLETEPVEDGKLGERGHEEGFLNNSGEFLFNKQLESIGIPQFHSPVGSPLKSIQATLTPSAMKSSPQIPHQTYSSILKHLN*. The sequence was codon-optimized by GenScript, and the restriction enzyme sites of the gene sequence are EcoRI and HindIII. The pET-30a plasmid was digested with EcoRI and HindIII to form a linear DNA fragment with sticky ends. The plasmid digestion reaction system is shown in Table 2. The gene sequence with restriction enzyme sites and the digested plasmid were ligated according to the ligation reaction system in Table 3.The ligation product was added to 50 μL of Trans-T1 competent cells and incubated on ice for 20 min; heat shocked at 42 °C for 90 s; incubated on ice for 2 min; 600 μL of LB liquid medium was added, and incubated at 37 °C at 110 rpm for 50 min; centrifuged at 2000 rpm for 3 min; 400 μL of the supernatant was discarded, the remaining part was mixed evenly, spread on the plate with a sterilized spreading rod, and incubated overnight at 38 °C; 10 colonies were picked, after the bacterial liquid was mixed evenly, sent to Tsingke for sequencing, and the sequencing results were analyzed with Snap software.

[0089] Table 2 Plasmid digestion reaction system

[0090]

[0091]

[0092] Table 3 Ligation system

[0093]

[0094] (2) Preparation of MAPK6 purified protein: Select the positive result bacterial liquid and extract the plasmid according to the plasmid extraction kit instructions. Then transfer it into BL21(DE3) competent cells. Take a 5000 mL baffled Erlenmeyer flask, add 2000 mL of LB liquid medium, add 2 mL of 50 mg / mL Kan antibiotic, shake well, inoculate 20 mL of bacterial liquid, shake the bacteria at 37 °C at 200 rpm. When OD600 reaches about 1.2, add IPTG inducer, the final concentration of IPTG is 0.2 mM, induce at 15 °C at 200 rpm for 16 h, centrifuge at 4500 rpm at 4 °C for 20 min, discard the supernatant, collect the bacterial liquid, resuspend the cell pellet with lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 10% Glycerol, pH 8.0), and then sonicate. The subsequent precipitate was centrifuged and dissolved with a denaturing agent.

[0095] Take 3 mL of beads and put them into the protein purification column, let it stand for 10 min. When the liquid level remains unchanged, drain 20% ethanol in the column, ddH 2 O was balanced 3 times, lysis buffer was balanced 3 times, and 15 mM imidazole was balanced 3 times. The crude protein supernatant was combined with the balanced beads and bound at 4 °C for 60 min. Elution was carried out successively with 15 mM and 50 mM imidazole. The G250 Coomassie brilliant blue solution was reacted with the collected eluate. When the G250 Coomassie brilliant blue solution did not change color, the miscellaneous proteins were completely eluted; the target protein was eluted with 150 mM imidazole solution, and SDS-PAGE was used to monitor the protein expression.

[0096] The protein concentration was about 1.36 mg / mL, centrifuged at 12000 rpm at 4 °C for 20 min, and the supernatant was aspirated into a new 1.5 mL centrifuge tube. ddH2 Elute with 30 mL of O, then wash with 30 mL of PBS; wash the injection loop with water and then with buffer; aspirate to draw the sample into the injection loop (be careful not to aspirate air); change the pump path to inject, and when 2 mL has been eluted, change the pump path to load and elute a volume of 25 mL; flow rate: 0.4 mL / min, pressure: 2 Mpa. Collect the sample according to the peaks.

[0097] (3) Detection of the interaction between DOX and MAPK6 by surface plasmon resonance technology: Dilute MAPK6 protein to 10 μg / ml with sodium acetate at pH 5.5, 5.0, 4.5, and 4.0 respectively, and prepare 200 μL of each. Through a pre-enrichment experiment, it was determined that the optimal coupling condition for MAPK6 protein is sodium acetate at pH 4.0. Then use an amino coupling kit to couple the positively charged MAPK6 protein with the -COOH groups on the activated chip surface, and the final coupled protein is 13,000 RU. Dilute 52.5 mL of 10×PBS-P to 500 mL with deionized water to prepare 1.05×PBS-P. Then, prepare calibration curve solvents of 4.5% and 5.8% DMSO using 1×PBS-P and pure DMSO respectively. Weigh 2.00 mg of DOX and add 5% DMSO to prepare a 20 mM DOX stock solution. Take 50 μL of 20 mM DOX and add it to 950 μL of 5% DMSO to obtain a 1 mM DOX stock solution. Then dilute the 1 mM DOX concentration to 50 μM with the prepared 5% DMSO running buffer as the highest injection concentration, and dilute it 1:1 downward by at least 5 concentration gradients (25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM). Set a repeated concentration at intervals and add a 0 concentration. Inject 0 μM, 0.78125 μM, 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM DOX, and the instrument records the change in SPR signal over time. Open the data analysis software BiacoreS200 Evaluation Software (T2002.0, GE Healthcare), click open in Data to find the file. Click on the one under Evaluation. Then select the sample to be analyzed, click next, and in Included Curves, you can select the sample concentrations to be analyzed. Then click Fit in the upper left corner to obtain the fitting result.

[0098] The results of SDS-PAGE detection of nickel column-purified MAPK6 protein are as Figure 3As shown in A, there are a few impurity proteins in the purified protein. The interaction of the MAPK6 protein purified by molecular sieve was detected and analyzed, and the interaction between DOX and MAPK6 was detected by surface plasmon resonance technology. As the concentration of DOX increased, the binding to the MAPK6 protein increased, enhancing the stability. After Affinity fitting, the KD was 9.2X10 -6 M, and DOX had a strong affinity for MAPK6 (as Figure 3 shown in B).

[0099] Example 4 Shengmai Injection Inhibits MAPK6 Expression and Reduces Apoptosis of Cardiomyocytes

[0100] The source of the Shengmai Injection used in this example: China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd., batch number 210602CK01.

[0101] Seventy-five 6- to 8-week-old male C57 / BL mice (20±2 g) were placed in a SPF environment for 3 days of adaptive feeding. All animal experiments were approved by the Animal Ethics Committee of the Medical Experiment Center of the China Academy of Chinese Medical Sciences (approval number: ERCCACMS21-2208-04). The mice were randomly divided into a Ctrl group, a Model group (5 mg / kg DOX), and a Model+SMI group (Model+SMI-L: 2.6 mL / kg SMI; Model+SMI-M: 5.2 mL / kg SMI, clinical equivalent dose; Model+SMI-H: 10.4 mL / kg SMI) (n = 15 / group).

[0102] Injection was administered intraperitoneally according to a volume of 0.1 mL per 10 g of mouse weight. Model mice were intraperitoneally injected with DOX once a week for 3 consecutive weeks. Mice in the SMI group were intraperitoneally injected for 28 days. The Ctrl group was given an equal volume of normal saline for 28 days.

[0103] The WB method was used to detect the expression levels of proteins such as MAPK6 and BAX in the heart tissue. The qPCR method was used to detect the mRNA expression levels of genes such as MAPK6, BAX, and NRF2 in the heart tissue. The qPCR primer list is shown in Table 4. The apoptosis of cardiomyocytes in the heart tissue was measured with reference to the instructions of the Tunel kit from Abbkine Company.

[0104] Table 4 GAPDH, BAX, MAPK6, NRF2 qPCR Primer List

[0105]

[0106] The WB and qPCR methods were respectively used to detect the expression levels of MAPK6 protein and mRNA in the heart tissue. The results are as Figure 4As shown in A, B, and C, compared with the DOX group, Shengmai injection at different concentrations could significantly inhibit the expression levels of MAPK6 protein and mRNA. The apoptosis of cardiomyocytes in the heart tissue was measured using a Tunel kit, and the results are as shown in Figure 4 D and E. Shengmai injection at different concentrations could significantly inhibit the apoptosis of cardiomyocytes in the heart tissue. The expression level of BAX in the heart tissue was detected by WB, and Shengmai injection could significantly inhibit the expression of BAX protein. The results are as shown in Figure 4 F and G. The mRNA expression level of BAX gene in the heart tissue was detected by qPCR. The expression level of BAX increased significantly in DOX, and Shengmai injection at different concentrations could significantly inhibit the expression level of BAX mRNA. The results are as shown in Figure 4 H.

[0107] The expression level of NRF2 decreased significantly in DOX, and Shengmai injection at different concentrations could significantly inhibit the expression level of NRF2 mRNA. The results are as shown in Figure 5 shown. Echocardiography showed that Shengmai injection at different concentrations could improve cardiac function (as shown in Figure 6 ), significantly increase EF% (as shown in Figure 7 ), and FS% (as shown in Figure 8 ).

[0108] From the above examples, it can be seen that:

[0109] 1. The present invention constructed a MAPK6 overexpression vector, and lentivirus coated with this vector was used to infect AC16 cells. The results showed that overexpression of MAPK6 could promote the expression of apoptosis factors in AC16 cells, increase the intracellular ROS level, and increase the expression level of DRP1 protein.

[0110] 2. The present invention designed 1 shRNA sequence and constructed a MAPK6 knockdown vector. The results showed that the shRNA sequence could effectively reduce the expression level of MAPK6 protein, inhibit the expression level of apoptosis gene mRNA, reduce the apoptosis of AC16 cells induced by DOX, and play an anti-apoptotic role in cardiomyocytes.

[0111] 3. The present patent also used surface plasmon resonance technology and found that DOX could have a strong affinity with MAPK6.

[0112] 4. By constructing an in vivo simulation of DOX cardiotoxicity, it was found that Shengmai powder injection could significantly inhibit the expression levels of MAPK protein and mRNA, inhibit the expression of apoptosis factors, reduce cardiomyocyte apoptosis, inhibit the expression of NRF2 mRNA, and improve cardiac function.

[0113] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of MAPK6 inhibitors in the preparation of drugs for the treatment of doxorubicin-induced cardiotoxicity.

2. The use according to claim 1, characterized in that The doxorubicin cardiotoxicity refers to the apoptosis of myocardial cells caused by doxorubicin.

3. The use according to claim 1, characterized in that The MAPK6 inhibitor is shRNA that inhibits the expression of MAPK6.

4. The use according to claim 3, characterized in that The nucleotide sequence of the shRNA is shown in SEQ ID NO:

1.

5. A drug for treating doxorubicin cardiotoxicity for use according to any one of claims 1 to 4, characterized in that: The drug is a biological preparation containing shRNA that inhibits the expression of MAPK6, and the nucleotide sequence of the shRNA is shown in SEQ ID NO:

1.

6. The drug for treating doxorubicin cardiotoxicity according to claim 5, characterized in that: The dosage forms of the biological preparation include injection preparations, powders and oral liquid preparations.

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