Application of oligopeptide MPM in preparation of reagent or medicine for treating liver injury and heart injury

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of a short peptide MPM in preparation of a reagent or a medicine for treating liver injury and heart injury. The amino acid sequence of the short peptide MPM is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 3. The oligopeptide MPM and the reagent for promoting the content of the oligopeptide MPM can be used for treating liver injury and heart injury, and particularly can be used for treating acetaminophen-induced liver injury and adriamycin-induced heart injury.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to the use of a short peptide MPM in the preparation of reagents or drugs for the treatment of liver and heart damage. Background Technology

[0002] The liver, a vital detoxification organ, is responsible for metabolizing and eliminating substances such as drugs, alcohol, organic solvents, chemicals, endogenous metabolites, and heme. When these substances are present in excessive amounts, the liver struggles to cope, leading to liver damage. Drugs are something almost everyone encounters in daily life. Drug-induced liver injury is one of the most common types of liver injury, especially prevalent among the elderly and those taking long-term medications. With the increasing use of modern drugs, particularly the combined use of multiple drugs and the overuse of herbal remedies and dietary supplements, the incidence of drug-induced liver injury is gradually rising. Drug-induced liver injury has become one of the leading causes of acute liver failure. Furthermore, drug-induced liver injury can not only lead to acute liver failure but also potentially cause long-term liver dysfunction, cirrhosis, and even liver cancer.

[0003] Because the symptoms of drug-induced liver injury are diverse and similar to those of other liver diseases (such as viral hepatitis and fatty liver), diagnosis is complex and may lead to delays in treatment. Currently, there are no specific drugs for the treatment of drug-induced liver injury; treatment mainly relies on discontinuation of the drug and supportive care.

[0004] Acetaminophen, abbreviated as APAP, is a commonly used antipyretic and analgesic drug that almost everyone comes into contact with. However, unintentional combined use or overdose of acetaminophen can induce acute liver damage and even liver failure. Acetaminophen is the leading cause of acute liver failure.

[0005] N-acetylcysteine ​​(NAC) is currently the only drug approved by the FDA for the treatment of acetaminophen-related liver injury. However, the therapeutic effect of NAC is highly dependent on early administration, has a therapeutic window, and may cause side effects including allergic reactions and fluid overload. Standard doses may be insufficient to treat patients with acetaminophen overdose. Therefore, finding a more effective therapeutic target remains an urgent problem to be solved.

[0006] Malignant tumors and cardiovascular diseases are the two major categories of diseases that threaten human health. Thanks to advancements in modern cancer treatment methods, the five-year survival rate for cancer patients has significantly improved. However, with the extension of patient survival, the impact of cancer treatment-related cardiotoxicity on patients is becoming increasingly prominent. This not only seriously affects patients' quality of life but also greatly limits their drug choices; heart failure caused by anti-tumor drugs can severely restrict patients' survival. In recent years, researching the potential mechanisms and prevention measures of anti-tumor drug cardiotoxicity has become a key focus and challenge in the fields of tumor pharmacology and oncological cardiology.

[0007] Currently, the main approach to preventing cardiotoxicity from antitumor drugs is considered to be reducing oxidative stress damage, such as through the use of antioxidants like vitamin E and N-acetylcysteine. However, while this strategy has shown initial success in animal models, it has proven ineffective in clinical trials, and the specific mechanisms remain unclear. The iron chelator dextromethorphan is currently the only FDA-approved and clinically recognized drug for preventing anthracycline-induced cardiotoxicity, primarily used in breast cancer patients, and has shown some preventative effects. However, dextromethorphan has certain adverse reactions, such as severe bone marrow suppression, and its use requires strict adherence to guidelines. Furthermore, animal studies have shown that erythropoietin and resveratrol can also reduce anthracycline-induced cardiotoxicity to some extent, but clinical data to support this are lacking.

[0008] Therefore, there is an urgent need to develop new prevention and treatment strategies for liver damage caused by excessive acetaminophen use and heart damage caused by anti-tumor treatment. Summary of the Invention

[0009] The purpose of this application is to overcome the shortcomings of the prior art and provide the application of a short peptide MPM in the preparation of reagents or drugs for treating liver and heart damage.

[0010] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0011] This application provides the use of an active substance in the preparation of a reagent or drug for treating liver injury and heart injury, said active substance comprising: a short peptide MPM, a nucleotide encoding the short peptide MPM, an expression vector containing the nucleotide encoding the short peptide MPM, and a nucleic acid inhibitor that increases the level of the short peptide MPM.

[0012] The nucleic acid inhibitor is a ribozyme, antisense RNA, or oligonucleotide inhibitor that specifically binds to microRNA-17-5p, microRNA-130a-3p, and / or microRNA-130b-3p.

[0013] Preferably, the short peptide MPM comprises a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO: 1 to 3.

[0014] Preferably, the nucleotide sequence encoding the short peptide MPM is as shown in any one of SEQ ID NO: 4 to 6;

[0015] The expression vector comprises a sequence as shown in any one of SEQ ID NO: 4 to 6;

[0016] The nucleic acid inhibitor comprises RNA with a sequence as shown in any one of SEQ ID NO: 7 to 9.

[0017] Specifically, the amino acid sequence of the short peptide MPM (human) is as follows: Madvsertlqlsvlvafasgvllgwqanrlrrryldwrkrrlqdklaatqkkldla (e.g., SEQ ID NO: 1).

[0018] The amino acid sequence of the short peptide MPM (mouse) is as follows:

[0019] Madvsertlqvsvlvafasgvvlgwqanrlrrryldwrkrrlqdklattqkkldla (eg SEQ ID NO: 2).

[0020] The amino acid sequence of the short peptide MPM(rat) is as follows:

[0021] MADVSERTLQVSVLVAFASGVVLGWQANRLRRRYLDWRKRRLQDKLA TTQKKLDLA (such as SEQ ID NO: 3).

[0022] The nucleotide sequence encoding the short peptide MPM (human) is as follows:

[0023] ATGGCGGATGTGTCAGAGAGGACACTGCAGTTGTCCGTGCTAGTAGC

[0024] CTTCGCTTCTGGAGTACTCCTGGGCTGGCAGGCGAACCGACTGCGGAGGC

[0025] GCTACTTGGACTGGAGGAAAAGGAGGCTGCAGGACAAGCTGGCGGCGAC

[0026] GCAGAAGAAGCTGGACCTGGCCTGA (SEQ ID NO: 4).

[0027] The nucleotide sequence encoding the short peptide MPM (mouse) is as follows:

[0028] ATGGCGGACGTGTCTGAGAGGACGCTGCAGGTGTCCGTGCTAGTGGC

[0029] TTTCGCCTCTGGAGTGGTCCTGGGCTGGCAAGCGAATCGGCTGCGGAGGC

[0030] GTTACCTAGACTGGAGGAAGCGGAGGCTGCAGGACAAGCTGGCAACGAC

[0031] TCAGAAAAGCTGGACCTGGCCTGA (SEQ ID NO: 5).

[0032] The nucleotide sequence encoding the short peptide MPM(rat) is as follows:

[0033] ATGGCGGACGTGTCTGAGAGGACGCTGCAAGTGTCCGTGCTAGTGGC

[0034] TTTCGCCTCTGGAGTGGTCCTTGGCTGGCAAGCGAATCGGTTGCGGAGGC

[0035] GTTACCTGGACTGGAGGAAGCGGAGGCTGCAGGACAAGCTGGCGACGAC

[0036] TCAGAAAAGCTGGACCTGGCCTGA (SEQ ID NO: 6).

[0037] Antisense RNAs that specifically bind to microRNA-17-5p, microRNA-130a-3p, and / or microRNA-130b-3p:

[0038] anti-miR-17-5p: CUACCUGCACUGUAAGCACUUUG (SEQ ID NO: 7);

[0039] anti-miR-130a-3p: AUGCCCUUUUAACAUUGCACUG (SEQ ID NO: 8); anti-miR-130b-3p: AUGCCCUUUCAUCAUUGCACUG (SEQ ID NO: 9).

[0040] Extensive experiments have shown that this application has discovered that the use of short peptide MPM can better protect the liver and heart, and treat liver and heart damage.

[0041] As a preferred embodiment of the application described in this application, the administration method of the short peptide MPM includes administration via liposomes.

[0042] As a preferred embodiment of the application described in this application, the method for preparing the liposomes includes the following steps:

[0043] 1) Weigh out lecithin, cholesterol, and distearate phosphatidylethanolamine-PEG2000-galactosamine and dissolve them in a chloroform / methanol mixture to obtain the dissolved product;

[0044] 2) The solution from step 1) is subjected to rotary evaporation until a uniform lipid film is formed, then nitrogen gas is introduced and the film is dried.

[0045] 3) Dissolve the MPM peptide in PBS and mix it with the dried homogeneous lipid membrane to obtain liposomes encapsulating the MPM peptide.

[0046] In some specific embodiments, the mass ratio of lecithin, cholesterol, and distearate phosphatidylethanolamine-PEG2000-trigalactosamine is 5:1:5.

[0047] In some specific embodiments, the mass ratio of chloroform to methanol is 1:1.

[0048] As a preferred embodiment of the application described in this application, the liposomes are used in animal experiments at a dosage of 5 mg / kg.

[0049] As a preferred embodiment of the application described in this application, the short peptide MPM includes a polypeptide containing the amino acid sequence of the short peptide MPM, such as an MPM polypeptide (MPM-FITC) with fluorescein isothiocyanate linked to its end.

[0050] As a preferred embodiment of the application described in this application, the liver injury includes liver injury induced by taking acetaminophen.

[0051] Analysis of public databases revealed that MPM levels were significantly reduced in the liver and hepatocyte-like cells affected by acetaminophen-induced liver injury, indicating that the short peptide MPM plays a role in acetaminophen-induced liver injury. Furthermore, in a mouse model of acetaminophen-induced liver injury, a single injection of MPM-FITC liposomes alleviated acetaminophen-induced liver damage; in a cell model of acetaminophen-induced liver injury, overexpression of MPM reduced the decrease in hepatocyte viability induced by acetaminophen. These findings suggest that the short peptide MPM, the nucleotide encoding the short peptide MPM, and expression vectors containing the nucleotide encoding the short peptide MPM can effectively treat acetaminophen-induced liver injury.

[0052] As a preferred embodiment of the application described in this application, the cardiac injury includes cardiac injury induced by chemotherapy drugs.

[0053] In a preferred embodiment of the application described in this application, the chemotherapy drug includes anthracyclines.

[0054] As a preferred embodiment of the application described in this application, the anthracycline drug includes doxorubicin.

[0055] Experiments showed that animals receiving doxorubicin chemotherapy developed cardiac damage, with a significant decrease in both the RNA and protein levels of MPM in the heart, indicating that the short peptide MPM plays a role in doxorubicin-induced cardiac damage. Further overexpression of MPM alleviated cardiomyocyte damage. This demonstrates that the short peptide MPM, the nucleotide encoding the short peptide MPM, and expression vectors containing the nucleotide encoding the short peptide MPM can treat doxorubicin-induced cardiac damage.

[0056] In the technical solution of this application, the short peptide MPM of this application can better protect the liver and heart, especially better treat acetaminophen-induced liver damage and doxorubicin-induced heart damage.

[0057] Compared with the prior art, this application has the following beneficial effects:

[0058] This application provides the use of a short peptide MPM in the preparation of reagents or drugs for treating liver and heart damage. The short peptide MPM of this application can better protect the liver and heart, especially better treat acetaminophen-induced liver damage and doxorubicin-induced heart damage. Attached Figure Description

[0059] Figure 1 The figure shows the results of significantly reduced levels of the short peptide MPM in the liver and hepatocyte-like cells of patients with acetaminophen-induced liver failure.

[0060] Figure 2 Following injection of a lethal dose (400 mg / kg) of acetaminophen, wild-type mice (MPM) f / f ) and hepatocyte-specific knockout MPM mice (MPM LKO Survival curves;

[0061] Figure 3 MPM f / f and MPM LKO Liver damage in mice after intraperitoneal injection of 200 mg / kg acetaminophen at different time points (0, 3, 6, 12, 18, 24, 48, 96 hours). Figure 3 -A is the MPM diagram. f / f and MPM LKOHematoxylin and eosin staining of mouse liver sections; Figure 3 -B diagram shows MPM f / f and MPM LKO Statistics on the area of ​​liver damage in mice; Figure 3 -C diagram is for MPM f / f and MPM LKO Mouse serum ALT and AST levels; Figure 3 -D diagram is for MPM f / f and MPM LKO Levels of inflammatory factors TNFα and IL-6 in mouse serum;

[0062] Figure 4 The therapeutic effect of MPM-FITC liposomes in a mouse model of acetaminophen-induced liver injury is shown in the figure. Figure 4 -A shows the hematoxylin and eosin staining of mouse liver sections and the statistical results of liver damage area after treatment with control (FITC) and MPM-FITC liposomes; Figure 4 -B represents the serum ALT and AST levels in mice after treatment with control (FITC) and MPM-FITC liposomes.

[0063] Figure 5 In a cell model of acetaminophen-induced liver injury, knockout or knockdown of MPM exacerbated the acetaminophen-induced decrease in hepatocyte viability, while overexpression of MPM alleviated the acetaminophen-induced decrease in hepatocyte viability. Figure 5 -A represents acetaminophen-treated wild-type (MPM) f / f ) and knockout MPM (MPM) LKO The viability of hepatocytes in each group was determined after primary mouse hepatocytes, control hepatocytes, or primary mouse hepatocytes overexpressing MPM. Figure 5 -B represents the cell viability of each group after treatment with acetaminophen on AML12 hepatocytes with stable MPM knockdown (shMPM) and their control cells (shNC), and AML12 hepatocytes with stable MPM overexpression (MPM) and their control cells (PCDH).

[0064] Figure 6 Figure showing the results of reduced cardiac MPM levels in a mouse model of doxorubicin-induced cardiac injury. Figure 6 -A represents the reduction of MPM RNA levels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) by doxorubicin treatment; Figure 6 -B is a schematic diagram of an experiment involving doxorubicin-induced cardiac injury in tumor-bearing mice; Figure 6 -C to Figure 6 -D indicates that the RNA and protein levels of MPM in the heart tissue of tumor-bearing mice are close to those in normal mice; Figure 6 -E indicates that mice experienced a decrease in body weight after treatment with doxorubicin; Figure 6 -F indicates that after treatment with doxorubicin, the serum lactate dehydrogenase (LDH) level in mice increased; Figure 6 -G to Figure 6 -H represents the decrease in RNA and protein levels of MPM in the heart tissue of tumor-bearing mice after doxorubicin treatment;

[0065] Figure 7 Figure showing the results of reduced MPM levels in a cardiomyocyte model of doxorubicin-induced cardiac injury. Figure 7 -A represents the reduction of MPM RNA levels in H9c2 cardiomyocytes after 6 hours of doxorubicin treatment; Figure 7 -B indicates that doxorubicin treatment for 12 and 24 hours reduces MPM protein levels in H9c2 cardiomyocytes; Figure 7 -C represents the reduction of H9c2 cardiomyocyte viability after 12 and 24 hours of doxorubicin treatment;

[0066] Figure 8 Figure showing the effect of knocking out MPM in a mouse model of doxorubicin-induced cardiac injury, exacerbating the doxorubicin-induced cardiac injury. Figure 8 -A represents the MPM in a model of doxorubicin-induced cardiac injury. + / + and MPM - / - Survival curves of mice; Figure 8 -B represents the MPM in the doxorubicin-induced cardiac injury model. + / + and MPM - / - Sirius red staining results of mouse heart tissue sections. The left and middle images are representative images of Sirius red staining in heart tissue, and the right image shows the proportion of Sirius red positive area to the total statistical area.

[0067] Figure 9 To exacerbate doxorubicin-induced cardiomyocyte apoptosis by knocking down MPM, the results of overexpressing MPM to weaken doxorubicin-induced cardiomyocyte apoptosis are shown in the figure. Figure 9 -A shows the expression of the apoptosis marker protein Caspase3 in control cardiomyocytes (RNAiMAX, si-GFP) and MPM-knockdown cardiomyocytes (si-MPM-1, si-MPM-2) after treatment with DOX; Figure 9 -B represents the expression of the apoptosis marker protein Caspase3 in control cells and MPM-overexpressing cells after DOX treatment;

[0068] Figure 10 The graph shows the increase in MPM protein levels after transfection with antisense RNA that specifically binds to microRNA-17-5p or microRNA-130a-3p. Figure 10-A indicates that MPM protein levels increased after transfection with antisense RNA anti-miR-17-5p of microRNA-17-5p; Figure 10 -B indicates that MPM protein levels increased after transfection with the antisense RNA anti-miR-130a-3p of microRNA-130a-3p. Detailed Implementation

[0069] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0070] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified, and the raw materials used in each parallel experiment are the same.

[0071] In the following embodiments:

[0072] The amino acid sequence of the short peptide MPM (human) is as follows: Madvsertlqlsvlvafasgvllgwqanrlrrryldwrkrrlqdklaatqkkldla (e.g., SEQ ID NO: 1).

[0073] The amino acid sequence of the short peptide MPM (mouse) is as follows:

[0074] Madvsertlqvsvlvafasgvvlgwqanrlrrryldwrkrrlqdklattqkkldla (eg SEQ ID NO: 2).

[0075] The amino acid sequence of the short peptide MPM(rat) is as follows:

[0076] MADVSERTLQVSVLVAFASGVVLGWQANRLRRRYLDWRKRRLQDKLATTQKKLDLA (eg SEQ ID NO: 3).

[0077] The nucleotide sequence encoding the short peptide MPM (human) is as follows:

[0078] ATGGCGGATGTGTCAGAGAGGACACTGCAGTTGTCCGTGCTAGTAGCCTTCGCTTCTGGAGTACTCCTGGGCTGGCAGGCGAACCGACTGCGGAGGCGCTACTTGGACTGGAGGAAAAGGAGGCTGCAGGACAAGCTGGCGGCGACGCAGAAGAAGCTGGACCTGGCCTGA(SEQ ID NO:4).

[0079] The nucleotide sequence encoding the short peptide MPM (mouse) is as follows:

[0080] ATGGCGGACGTGTCTGAGAGGACGCTGCAGGTGTCCGTGCTAGTGGCTTTCGCCTCTGGAGTGGTCCTGGGCTGGCAAGCGAATCGGCTGCGGAGGCGTTACCTAGACTGGAGGAAGCGGAGGCTGCAGGACAAGCTGGCAACGACTCAGAAAAAGCTGGACCTGGCCTGA(SEQ ID NO:5).

[0081] The nucleotide sequence encoding the short peptide MPM (rat) is as follows:

[0082] ATGGCGGACGTGTCTGAGAGGACGCTGCAAGTGTCCGTGCTAGTGGCTTTCGCCTCTGGAGTGGTCCTTGGCTGGCAAGCGAATCGGTTGCGGAGGCGTTACCTGGACTGGAGGAAGCGGAGGCTGCAGGACAAGCTGGCGACGACTCAGAAAAAGCTGGACCTGGCCTGA(SEQ ID NO:6).

[0083] RNA sequence:

[0084] anti-miR-17-5p: CUACCUGCACUGUAAGCACUUUG(SEQ ID NO:7);

[0085] anti-miR-130a-3p: AUGCCCUUUUAACAUUGCACUG(SEQ ID NO:8);

[0086] anti-miR-130b-3p: AUGCCCUUUCAUCAUUGCACUG(SEQ ID NO:9).

[0087] Example 1

[0088] The levels of the short peptide MPM were significantly reduced in the livers of patients with acetaminophen-induced liver failure and in human hepatocyte-like cells.

[0089] Specific experiment:

[0090] Figure 1 -A, Liver samples from healthy individuals were taken from individuals who died from brain injury (Ctrl, n=2), and liver samples from patients with acute liver failure (ALF) were taken from patients who developed ALF due to an overdose of acetaminophen (APAP) (n=3). Data were obtained from the GEO database (GSE74000), and the RNA levels of MPM were analyzed in the above data.

[0091] Figure 1 -B, Control (Ctrl) and acetaminophen (APAP) were used to analyze the RNA levels of MPM in hSKP-HPC cells (human skin precursor-derived hepatocyte-like cells).

[0092] Example 2

[0093] In a mouse model of liver failure induced by high-dose acetaminophen, hepatocyte-specific knockout MPM mice had shorter survival times.

[0094] Specific experiment:

[0095] Hepatocyte-specific knockout MPM mice (denoted as MPM) LKO Mice) and control mice (denoted as MPM) f / f Mice).

[0096] in:

[0097] MPM LKO The method for constructing mice includes the following steps:

[0098] Mice with hepatocyte-specific Cre (Alb-Cre) expression were compared with MPM. fl / fl Mouse [MPM sequence surrounded by loxP site (MPM)] fl / fl Mice (purchased from Jicui Pharmaceutical Co., Ltd.) were mated to obtain offspring (F1). F1 mice were then interbred. In the F2 generation, genotyping was used to select mice with the following genotypes: Alb-Cre; MPM. fl / fl Mice specifically knocking out MPM in hepatocytes, which are referred to as MPM in this application. LKO Mice; genotype (-); MPM fl / fl The mice in this study were those without Cre expression, and their MPM was preserved. These mice were used as control mice and denoted as MPM. f / f Mice.

[0099] MPM f / f Mice and MPM LKO Mice were fasted overnight (16 hours) and then intraperitoneally injected with a lethal dose of acetaminophen (400 mg / kg), with their condition monitored in real time. Results showed that after injection of the lethal dose of APAP, compared with MPM... f / f Compared to mice, MPM LKO The survival rate of mice was even lower.

[0100] Example 3

[0101] In a mouse model of liver injury induced by low-dose acetaminophen, MPM LKO The liver damage in mice was aggravated.

[0102] The specific experiment is as follows:

[0103] The method for constructing a mouse model of acetaminophen-induced liver injury includes the following steps:

[0104] MPM f / f and MPM LKO Mice were fasted overnight (16 hours) and then injected intraperitoneally with 200 mg / kg acetaminophen. The mice were collected at different time points (0, 3, 6, 12, 18, 24, 48 and 96 hours later).

[0105] like Figure 3 As shown, in the acetaminophen-induced liver injury model, MPM LKO The liver damage was more severe in the mice.

[0106] in, Figure 3 -A represents MPM f / f and MPM LKO Hematoxylin and eosin staining of mouse liver sections.

[0107] Figure 3 -B stands for MPM f / f and MPM LKO Statistics on the area of ​​liver damage in mice.

[0108] Figure 3 -C is for MPM f / f and MPM LKO Serum ALT and AST levels in mice.

[0109] Figure 3 -D stands for MPM f / f and MPM LKO The levels of inflammatory factors TNFα and IL-6 in mouse serum.

[0110] Example 4

[0111] In a mouse model of acetaminophen-induced liver injury, the liver injury was significantly alleviated after treatment with MPM-FITC.

[0112] The specific experiment is as follows:

[0113] Wild type (MPM) f / f One hour after injecting mice with 200 mg / kg acetaminophen, they were injected via the tail vein with 5 mg / kg of liposomes encapsulated with FITC or MPM-FITC. The mice were harvested 17 hours later.

[0114] The method for constructing liposomes of FITC and MPM-FITC includes the following steps:

[0115] 1. Weigh out lecithin (500mg), cholesterol (100mg), and DSPE-PEG2000-GalNAC (50mg) (purchased from Guangzhou Carbohydrate Biotechnology Co., Ltd.). Dissolve in 50ml of chloroform / methanol mixture for 5-10 minutes. (Lecithin:cholesterol:DSPE = 5:1:5, chloroform:methanol = 1:1)

[0116] 2. After the above reagents have completely dissolved, divide the solution into two equal bottles and evaporate them on a rotary evaporator (40℃, 200rpm) for about 0.5-1 hour until a uniform lipid film is formed.

[0117] 3. After evaporation is complete, fill the bottle with nitrogen gas for about 5 minutes.

[0118] 4. Place in a vacuum drying oven overnight to remove residual organic solvents.

[0119] 5. First, dissolve MPM-FITC (total amount 5mg, final concentration 2mg / mL) and FITC (0.2825mg required according to molecular weight conversion) in PBS, and add PBS to 25mL. Dissolve at room temperature for 2 hours, mixing once every half hour to ensure that the lipid membrane on the wall is completely immersed in PBS.

[0120] MPM-FITC is an MPM peptide followed by a FITC (fluorescein isothiocyanate).

[0121] The amino acid sequence of MPM (mouse) is as follows:

[0122] Madvsertlqvsvlvafasgvvlgwqanrlrrryldwrkrrlqdklattqkkldla (SEQ ID NO: 2).

[0123] Figure 4-A represents the statistical results of hematoxylin and eosin staining and liver injury area in mouse liver sections after injection of control (FITC) and MPM liposomes (MPM-FITC) in a mouse model of acetaminophen-induced liver injury.

[0124] Figure 4 Figure B shows the serum ALT and AST levels in mice after injection of control (FITC) and MPM liposomes (MPM-FITC) in a mouse model of acetaminophen-induced liver injury.

[0125] The above experiments show that MPM-FITC treatment significantly alleviated acetaminophen-induced liver damage.

[0126] Example 5

[0127] In a cell model of acetaminophen-induced liver injury, knockdown or knockout of MPM exacerbated hepatocellular damage, while overexpression of MPM alleviated hepatocellular damage.

[0128] The specific experiment is as follows:

[0129] Figure 5 -A Left Image, from MPM LKO Mice and MPM f / f Primary hepatocytes with MPM knockout and control primary hepatocytes were isolated from mice. After cell adhesion, the cells were treated with acetaminophen (5 mM) for 24 hours, and cell viability was detected using the CCK8 assay kit.

[0130] Figure 5 -A Right Figure, from MPM f / f Primary hepatocytes were isolated from mice. After cell adhesion, the control plasmid PCDH and the plasmid overexpressing MPM were transfected with lipofectamin 3000 (purchased from Thermo Fisher Scientific) for 24 hours. After treatment with acetaminophen (5 mM) for 24 hours, cell viability was detected using the CCK8 assay kit.

[0131] Figure 5 -B, Stable AML12 cell lines with stable MPM knockdown (denoted as shMPM) and their control cells (denoted as shNC), or stable AML12 cell lines with stable MPM overexpression (denoted as MPM) and their control cells (denoted as PCDH), were treated with acetaminophen (5mM) for 24 hours, and cell viability was detected using a CCK8 assay kit.

[0132] in,

[0133] The steps for constructing stable strains by knocking down or overexpressing MPM are as follows:

[0134] 1) First, prepare lentiviruses with knockdown of MPM, overexpression of MPM, or control.

[0135] Day 1: 293T cells were deposited in a 6-well plate, 200,000 cells / well.

[0136] Day 2: When the cell density is about 40%-50%, transfect the cells with lentiviral packaging plasmids (PMDIG, PSPAX2) and knockdown MPM (shMPM) plasmids or control plasmids (shNC), or overexpress MPM plasmids or control plasmids (PCDH). Change the medium after 6 hours.

[0137] Day 5: Collect the virus solution. Collect the culture supernatant of 293T cells into a 15mL centrifuge tube, centrifuge at 1000g for 10 minutes, transfer the supernatant to a new EP tube, and store at -80℃.

[0138] 2) Preparation of AML12 cells stably knocked down or overexpressing MPM and their control cells:

[0139] One day in advance (day 6), AML12 cells were attached to wells in 24-well plates at a density of 20,000 cells / well. On the following day (day 7), 200 μL of cell culture medium, 200 μL of virus solution, and 0.4 μL of polybrene (final concentration of 10 μg / mL; the virus solution was collected according to the steps above; polybrene was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) were added to each well. The medium was changed after 24 hours.

[0140] Day 8: Expand the cells to a 6cm dish.

[0141] Day 9: Expand the cells to a 10cm dish.

[0142] Day 10: Add puromycin to screen for positive cells.

[0143] Day 12: Obtain AML12 cells with stable knockdown or overexpression of MPM and their control cells.

[0144] Example 6

[0145] In a mouse model of doxorubicin-induced cardiac injury, cardiac MPM levels were reduced.

[0146] The specific experiment is as follows:

[0147] Doxorubicin-induced cardiac injury modeling experimental steps:

[0148] Eight-week-old wild-type male mice were subcutaneously inoculated with 300,000 Hepa1-6 cells in both hind legs. After 14 days, they were randomly divided into two groups: one group was injected intraperitoneally with doxorubicin (DOX, 25 mg / kg), and the other group was injected with an equal volume of PBS (control). The administration frequency was once every two days. The body weight of mice in the control and treatment groups was recorded at each administration. The mice were harvested after 7 days.

[0149] Figure 6 -A represents the reduction in MPM RNA levels in human induced pluripotent hepatocyte-derived cardiomyocytes (hiPSC-CMs) after doxorubicin treatment. GSE157282 data (high-throughput sequencing data of hiPSC-CMs after doxorubicin treatment) were analyzed. Figure 6 -B is a schematic diagram of the doxorubicin-induced cardiac injury modeling experiment in tumor-bearing mice. Figure 6 -C to Figure 6 -D represents the RNA and protein levels of MPM in the heart tissues of tumor-bearing and non-tumor-bearing mice; specifically, 300,000 Hepa1-6 cells or serum-free culture medium (Control) were subcutaneously injected into the hind legs of two 8-week-old male mice, and mouse heart tissue was collected 21 days later for qPCR ( Figure 6 -C) and immunoblotting assay ( Figure 6 -D). Figure 6 -E to Figure 6 -F indicates that after treatment with doxorubicin, tumor-bearing mice experienced a decrease in body weight and an increase in serum lactate dehydrogenase (LDH) levels. Figure 6 -G to Figure 6 -H indicates that after treatment with doxorubicin, the RNA and protein levels of MPM in the heart tissue of tumor-bearing mice decreased.

[0150] The results showed that, compared with normal mice, the level of MPM in the heart tissue of tumor-bearing mice did not change significantly. Figure 6 -C to Figure 6 -D), indicating that tumor-bearing status does not affect MPM levels in cardiac tissue. This application observed that after doxorubicin treatment, tumor-bearing mice experienced a decrease in body weight ( Figure 6 -E), serum lactate dehydrogenase (LDH) levels are elevated ( Figure 6 -F) indicates that the mice developed cardiac damage after receiving doxorubicin chemotherapy; at this time, the RNA and protein levels of MPM were significantly decreased. Figure 6 -G to Figure 6 -H) suggests that doxorubicin treatment leads to a decrease in MPM levels in cardiac tissue.

[0151] Example 7

[0152] MPM was downregulated early in doxorubicin-induced cardiomyocyte damage.

[0153] The specific experiment is as follows:

[0154] In this embodiment, H9c2 cells were treated with doxorubicin (1 μM), and RNA was collected after 6 hours for qPCR detection. Figure 7 -A), or collect the protein after 12 or 24 hours for immunoblotting. Figure 7 -B) or perform cell viability testing after 6, 12, or 24 hours ( Figure 7 -C).

[0155] like Figure 7 As shown, MPM is downregulated early in doxorubicin-induced cardiomyocyte injury.

[0156] The results showed that after 6 hours of treatment with doxorubicin, the RNA level of MPM in H9c2 cells decreased significantly. Figure 7 -A), while at this time the cell viability has not yet changed significantly ( Figure 7 -C); while in cells treated with doxorubicin for 12 and 24 hours, MPM protein levels and cell viability were significantly reduced (-C); Figure 7 -B to Figure 7 -C). This suggests that MPM RNA levels decrease in the early stages of cardiomyocyte injury, and that this decrease in RNA levels may lead to a reduction in MPM protein levels and decreased cell viability in the later stages of injury.

[0157] Example 8

[0158] Knockout of MPM exacerbates doxorubicin-induced cardiac damage.

[0159] The specific experiment is as follows:

[0160] This application involves wild-type mice (MPM) + / + ) and systemic knockout MPM mice (MPM - / - Intraperitoneal injection of doxorubicin (25 mg / kg) was used to induce acute cardiac injury. The effect of knocking out MPM on doxorubicin-induced acute cardiac injury was analyzed.

[0161] MPM- / - mice were bred in the C57BL / 6J genetic background and the open reading frame of the MPM gene was disrupted using the CRISPR-Cas9 system, resulting in MPM being untranslatable.

[0162] MPM at 18-19 weeks of age + / + and MPM - / - Mice were intraperitoneally injected with 25 mg / kg doxorubicin (DOX), and their survival status was observed every 24 hours. Survival curves were plotted. Figure 8 -A). Mouse heart tissue was collected and stained with Sirius red to indicate fibrotic areas ( Figure 8 -B). Figure 8 The left and middle images of -B are representative images of Sirius red staining in cardiac tissue, with a scale bar of 50 μm; Figure 8 The right-hand figure for -B shows the proportion of Sirius red positive area to the total statistical area.

[0163] The results showed that, compared with MPM + / + Compared to mice, MPM - / - Mice injected with doxorubicin died faster. Figure 8 -A). Sirius red staining of mouse hearts was used to assess the extent of cardiac damage. Results showed that after injection of doxorubicin, MPM... - / - Sirius red staining area is larger in mouse heart tissue ( Figure 8 -B), prompting MPM - / - The mice had a larger area of ​​cardiac fibrosis and more severe cardiac damage.

[0164] The above results preliminarily suggest that a decrease in MPM levels may exacerbate cardiac injury during doxorubicin-induced cardiac injury.

[0165] Example 9

[0166] Knockdown of MPM exacerbates doxorubicin-induced cardiomyocyte apoptosis, while overexpression of MPM attenuates doxorubicin-induced cardiomyocyte apoptosis.

[0167] The specific experiment is as follows:

[0168] Rat cardiomyocytes (H9c2) were transfected with RNAiMAX (Thermo Fisher Scientific) containing knockdown MPM siRNA (si-MPM-1 or si-MPM-2) or control siRNA (siGFP). After 24 hours, cells were treated with doxorubicin (1 μM), and cell lysate was collected for Western blotting. RNAiMAX was used as the RNAiMAX transfected control group, without siRNA transfection. The specific sequence of siMPM is as follows:

[0169] siMPM-1:

[0170] Forward primer:

[0171] GCUCGUCCUUGCACGGCUUdTdT (SEQ ID NO: 10);

[0172] Reverse primer:

[0173] AAGCCGUGCAAGGACGAGCdCdG (SEQ ID NO: 11).

[0174] si-MPM-2:

[0175] Forward primer:

[0176] GGAUAUAAAGCACGGGAAAdTdT (SEQ ID NO: 12);

[0177] Reverse primer:

[0178] UUUCCCGUGCUUUAUAUCCdAdT (SEQ ID NO: 13);

[0179] Experiments on overexpression of MPM:

[0180] On the first day, rat cardiomyocytes (H9c2) were applied at a density of approximately 20%. On the second day, pCDH-MPM (rat sequence) plasmid or control plasmid was transfected with lipo3000 liposomes. After 24 hours, doxorubicin (1 μM) was added. After 24 hours of treatment, cell lysate proteins were collected and Western blotting was performed to detect the expression of the apoptosis marker protein Caspase3.

[0181] The results showed that knocking down MPM aggravated doxorubicin-induced cardiomyocyte apoptosis, while overexpression of MPM attenuated doxorubicin-induced cardiomyocyte apoptosis.

[0182] Example 10

[0183] Mouse fibroblasts were transfected with anti-NC or antisense RNAs of microRNA-17-5p and microRNA-130a-3p (anti-miR-17-5p or anti-miR-130a-3p) using RNAiMAX (Thermo Fisher Scientific). Cell lysate proteins were collected and Western blotting was performed to detect the expression level of MPM.

[0184] Anti-NC serves as the control group for either anti-miR-17-5p or anti-miR-130a-3p; the specific sequences of anti-miR-17-5p or anti-miR-130a-3p are as follows:

[0185] anti-miR-17-5p: CUACCUGCACUGUAAGCACUUUG (SEQ ID NO: 7);

[0186] anti-miR-130a-3p: AUGCCCUUUUAACAUUGCACUG (SEQ ID NO: 8).

[0187] Transfection with antisense RNA that specifically binds to microRNA-17-5p or microRNA-130a-3p increased the protein level of MPM. Figure 10 -A indicates that MPM protein levels increase after transfection of microRNA-17-5p antisense RNA anti-miR-17-5p into fibroblasts. Figure 10 -B indicates that MPM protein levels increase after fibroblasts are transfected with the antisense RNA anti-miR-130a-3p of microRNA-130a-3p.

[0188] Since the sequences of microRNA-130a-3p and microRNA-130b-3p are very similar, differing by only one base, this application suggests that the antisense RNA of microRNA-130b-3p also has the function of increasing MPM protein levels.

[0189] In summary, the short peptide MPM of this application can protect the liver and heart, and MPM can be used to prepare reagents or drugs for treating liver and heart damage.

[0190] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. The use of an active substance in the preparation of a reagent or drug for treating liver and heart damage, characterized in that, The active substances include: short peptide MPM, nucleotides encoding short peptide MPM, expression vectors containing nucleotides encoding short peptide MPM, and nucleic acid inhibitors that increase the level of short peptide MPM. The nucleic acid inhibitor is a ribozyme, antisense RNA, or oligonucleotide inhibitor that specifically binds to microRNA-17-5p, microRNA-130a-3p, and / or microRNA-130b-3p.

2. The application as described in claim 1, characterized in that, The short peptide MPM contains a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO: 1 to 3.

3. The application as described in claim 1, characterized in that, The nucleotide sequence encoding the short peptide MPM is shown in any one of SEQ ID NO: 4 to 6; The expression vector comprises a sequence as shown in any one of SEQ ID NO: 4 to 6; The antisense RNA comprises RNA with a sequence as shown in any one of SEQ ID NO: 7 to 9.

4. The application as described in claim 1, characterized in that, The administration routes of the short peptide MPM include administration via liposomes.

5. The application as described in claim 1, characterized in that, The short peptide MPM includes an MPM polypeptide with fluorescein isothiocyanate linked to its terminal end.

6. The application as described in claim 1, characterized in that, The liver damage mentioned includes liver damage induced by taking acetaminophen.

7. The application as described in claim 1, characterized in that, The cardiac injury mentioned includes cardiac injury induced by chemotherapy drugs.

8. The application as described in claim 7, characterized in that, The chemotherapy drugs include anthracyclines.

9. The application as described in claim 8, characterized in that, The anthracycline drugs include doxorubicin.

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