Application and composition of substances targeting NUP35 gene and / or NUP35 protein

Abstract

This application relates to the field of biomedicine, and in particular to applications and compositions of substances targeting the NUP35 gene and / or the NUP35 protein. The substances targeting the NUP35 gene are promoters, or active polypeptides of recombinant NUP35 proteins or NUP35 protein fragments, that can increase NUP35 gene activity and / or expression. The substances targeting the NUP35 protein are promoters, or active polypeptides of recombinant NUP35 proteins or NUP35 protein fragments, that can increase NUP35 protein expression and function. The applications provided herein can be used to prepare compositions or preparations for treating cardiovascular diseases such as heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to applications and compositions of substances targeting the NUP35 gene and / or the NUP35 protein. Background Art

[0002] Nucleoporins (NUPs) are components of the nuclear pore complex (NPC) and are involved in nucleus / cytoplasm trafficking. Nuclear pore proteins are crucial for cell viability and gene transcriptional regulation. Nuclear pore protein 35 (NUP35) selectively regulates pH homeostasis in cultured cardiomyocytes by posttranscriptionally controlling Na(+)-H(+) exchanger-1 (NHE1) expression. Despite recent breakthroughs in reconstructing the molecular structure of the NPC using cryo-electron microscopy (cryo-EM), the role of NUP35 in heart failure remains unknown. Summary of the Invention

[0003] The purpose of the present invention is to provide a method and application of a drug using the NUP35 gene and / or NUP35 protein as a tissue cell-selective specific drug target in the treatment of angiotensin II and endothelial injury, myocardial infarction, ischemia-reperfusion, high-fat and high-fructose, and aging-induced heart failure.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] In one embodiment, a substance with the NUP35 gene as a drug target is used in the preparation of a drug for treating heart failure. The substance with the NUP35 gene as a drug target is a promoter that can increase the activity and / or expression level of the NUP35 gene, or a NUP35 recombinant protein or a NUP35 protein fragment active polypeptide.

[0006] Preferably, the nucleic acid sequence of the NUP35 gene is SEQ ID NO.2 or SEQ ID NO.4.

[0007] Preferably, the promoters capable of increasing the activity and / or expression level of the NUP35 gene include: small molecule compounds, polypeptides, and vectors for expressing NUP35.

[0008] In another embodiment, a substance with NUP35 protein as a drug target is used in the preparation of a drug for treating heart failure, wherein the drug with NUP35 protein as a drug target is a promoter that can increase the expression and function of NUP35 protein, or a NUP35 recombinant protein, or a NUP35 protein fragment active polypeptide, or a NUP35 protein fragment active polypeptide.

[0009] Preferably, the amino acid sequence of the NUP35 protein is SEQ ID NO.1 or SEQ ID NO.3.

[0010] Preferably, the promoters capable of improving the expression and function of NUP35 protein include: vectors, small molecule compounds, and polypeptides for constructing a NUP35 gene overexpression system.

[0011] Preferably, the NUP35 recombinant protein refers to a recombinant protein synthesized based on the amino acid sequence SEQ ID NO.1 or SEQ ID NO.3.

[0012] Preferably, the NUP35 recombinant protein includes: a modified NUP35 protein, a protein molecule that is homologous to a natural NUP35 protein and has the activity of a natural NUP35 protein, a dimer or multimer of a NUP35 protein, and an active polypeptide containing the amino acid sequence of a NUP35 protein.

[0013] More preferably, the modified NUP35 protein is a PEGylated NUP35 protein. Chemical derivatization of proteins in vivo or in vitro includes acetylation, carboxylation, and glycosylation.

[0014] More preferably, a protein molecule that is homologous to a natural NUP35 protein and has the activity of a natural NUP35 protein refers to a protein molecule whose amino acid sequence has 50% or more, preferably 60% or more, 70% or more, 80% or more, 85% or more, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% homology with SEQ ID NO.1 or SEQ ID NO.3.

[0015] More preferably, the above-mentioned vectors include: adenovirus, adeno-associated virus, lentivirus, plasmid, liposome and nanoparticles.

[0016] Preferably, the substance using NUP35 gene and / or NUP35 protein as a drug target can be selected from one or more of the following uses:

[0017] (1) Inhibit angiotensin II and heart failure after endothelial injury;

[0018] (2) inhibiting myocardial cell hypertrophy;

[0019] (3) inhibit cardiac cell fibrosis;

[0020] In another embodiment, the present invention provides a composition comprising a pharmaceutically acceptable carrier and a substance targeting the NUP35 gene and / or the NUP35 protein; wherein the NUP35 recombinant protein is a protein derivative synthesized based on the amino acid sequence SEQ ID NO.1 or SEQ ID NO.3.

[0021] Preferably, the above composition further comprises other components for treating heart failure.

[0022] The relevant definitions of the present invention are:

[0023] (1) NUP35 protein and its encoding nucleic acid

[0024] The present invention relates to a NUP35 protein and a truncated form thereof, wherein the amino acid sequence of the NUP35 protein is shown in SEQ ID NO. 1 or SEQ ID NO. 3. The NUP35 protein or a promoter thereof of the present invention can be used to: (1) inhibit angiotensin II and heart failure after endothelial injury; (2) inhibit myocardial cell hypertrophy; and (3) inhibit cardiac cell fibrosis.

[0025] The present invention also includes polypeptides or proteins with the same or similar functions that have 50% or more, preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, and most preferably 98% or more homology to the sequence shown in SEQ ID NO.1 or SEQ ID NO.3 of the present invention.

[0026] Among them, SEQ ID NO.1 is human NUP35 protein, and SEQ ID NO.3 is mouse NUP35 protein.

[0027] As used herein, the term "treat," when referring to protecting a mammal from a disease, means preventing, inhibiting, suppressing, or eliminating the disease. Preventing a disease includes administering a composition of the present invention to a mammal prior to the onset of the disease. Suppressing a disease includes administering a composition of the present invention to a mammal after induction of the disease but prior to clinical manifestation. Suppressing a disease includes administering a composition of the present invention to a mammal after clinical manifestation of the disease such that the disease is alleviated or maintained. Eliminating a disease includes administering a composition of the present invention to a mammal after clinical manifestation of the disease such that the mammal is no longer suffering from the disease.

[0028] The proteins of the present invention can be recombinant proteins, natural proteins, or synthetic proteins. The proteins of the present invention can be naturally purified products, or chemically synthesized products, or produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the proteins of the present invention can be glycosylated or non-glycosylated. The proteins of the present invention may or may not include an initial methionine residue.

[0029] The present invention also includes NUP35 protein fragments, active polypeptides and analogs having NUP35 protein activity. The terms "fragment" and "analog" used herein refer to proteins that substantially retain the same biological function or activity as the natural NUP35 protein of the present invention.

[0030] The mutant protein fragments, derivatives or analogs of the present invention can be: (1) mutant proteins having one or more conservative or non-conservative amino acid residues substituted, preferably conservative amino acid residues, and such substituted amino acid residues may or may not be encoded by the genetic code; or (2) mutant proteins having a substituent group in one or more amino acid residues; or (3) mutant proteins formed by fusion of a mature mutant protein with another compound, such as a compound that extends the half-life of the mutant protein (e.g., polyethylene glycol); or (4) mutant proteins formed by fusion of additional amino acid sequences to the mutant protein sequence, such as a leader sequence, a secretory sequence, a sequence for purifying the mutant protein or a proprotein sequence, and an antigen IgG fragment. According to the description herein, these fragments, derivatives and analogs are within the scope of those skilled in the art. In the present invention, conservatively substituted amino acids are preferably generated by amino acid substitution according to Table 1.

[0031] Table 1. Conservatively substituted amino acids

[0032] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile;Leu;Met;Phe;Ala Leu

[0033] The present invention also includes polypeptides or proteins with identical or similar functions that share 50% or greater, preferably 60% or greater, 70% or greater, 80% or greater, more preferably 90% or greater, even more preferably 95% or greater, and most preferably 98% or greater homology with the native NUP35 protein of the present invention. Protein fragments may include derivative sequences obtained by substitution, deletion, or addition of at least one amino acid (typically 1-60, preferably 1-30, more preferably 1-20, and most preferably 1-10), as well as the addition of one or more amino acids (typically within 20, preferably within 10, and more preferably within 5) to the C-terminus and / or N-terminus. For example, substitution of amino acids with similar or similar properties in the protein generally does not alter the protein's function, and the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the protein's function. The present invention includes analogs of natural NUP35 proteins that differ from natural NUP35 proteins in that they may be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced protein truncations can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known biological techniques. Analogs also include analogs with residues other than natural L-amino acids (such as D-amino acids), as well as analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

[0034] Modifications (which generally do not alter the primary structure) include chemical derivatization of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those that occur during protein synthesis and processing. Such modifications can be achieved by exposing the protein to glycosylation enzymes (e.g., mammalian glycosylases or deglycosylases). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Furthermore, modifications can be made to protein truncations of the present invention. Modifications (which generally do not alter the primary structure) include chemical derivatization of mutant proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those that occur during the synthesis and processing of the mutant protein, or in further processing steps. Such modifications can be achieved by exposing the mutant protein to glycosylation enzymes (e.g., mammalian glycosylases or deglycosylases). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Also included are muteins that have been modified to increase their resistance to proteolysis or optimize their solubility.

[0035] The present invention also provides polynucleotide sequences encoding NUP35 proteins. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include: DNA, genomic DNA, or synthetic DNA, and the DNA may be single-stranded or double-stranded. Polynucleotides encoding mature polypeptides include: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and non-coding sequences. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide or a polynucleotide further including additional coding and / or non-coding sequences. The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as the present invention. Variants of the polynucleotides may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded polypeptide.

[0036] In a preferred embodiment of the present invention, the polypeptide sequence of the NUP35 protein is shown as SEQ ID NO.1 or SEQ ID NO.3, and the polynucleotide sequence encoding the NUP35 protein is shown as SEQ ID NO.2 or SEQ ID NO.4.

[0037] Among them, SEQ ID NO.2 is the nucleotide sequence of the human NUP35 gene; SEQ ID NO.4 is the nucleotide sequence of the mouse NUP35 gene.

[0038] Based on the nucleotide sequences described herein, those skilled in the art can readily produce the encoding nucleic acids of the present invention using various known methods. These methods include, but are not limited to, PCR and DNA synthesis. For specific methods, see J. Sambrook, Molecular Cloning: A Laboratory Manual. As one embodiment of the present invention, the encoding nucleic acid sequences of the present invention can be constructed by synthesizing nucleotide sequences in segments followed by overlap extension PCR.

[0039] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0040] The proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0041] The full-length sequences of the polynucleotides of the present invention can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. For long sequences, two or more PCR amplifications are often required, followed by splicing the fragments amplified in the correct order.

[0042] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0043] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0044] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0045] Methods using PCR techniques to amplify DNA / RNA are preferably used to obtain the polynucleotides of the present invention. In particular, when full-length cDNA is difficult to obtain from a library, the RACE method (RACE - rapid amplification of cDNA ends) is preferably used. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods, such as gel electrophoresis.

[0046] (2) NUP35 promoter

[0047] In the present invention, the NUP35 promoter includes substances that can increase the activity and / or content of the NUP35 gene or its protein in vivo or in vitro.

[0048] Among them, the expression level of NUP35 can be increased by the following methods: the tissue itself secretes a large amount of NUP35 protein or artificially overexpresses NUP35 protein, or artificially delivers NUP35 protein (for example, using a viral vector, such as an adeno-associated virus vector) or NUP35 truncated active protein polypeptide, or NUP35 promoter.

[0049] In the present invention, the NUP35 promoter is not particularly limited, and any promoter that can promote the expression of NUP35 or enhance the activity of NUP35 protein is within the scope of protection of the present invention.

[0050] In a preferred embodiment, the NUP35 promoter comprises a small molecule compound.

[0051] (3) Compound pharmaceutical compositions and kits

[0052] The present invention provides a compound pharmaceutical composition containing an active ingredient and a pharmaceutically acceptable carrier. The active ingredient includes: a NUP35 gene promoter, a NUP35 truncated active protein polypeptide, or a NUP35 promoter; the carrier includes, but is not limited to: saline, buffer, glucose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should be compatible with the route of administration. The pharmaceutical composition of the present invention can be formulated as an injectable, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets, and capsules are preferably manufactured under sterile conditions. The pharmaceutical composition of the present invention can also be formulated as a powder for aerosol inhalation. The pharmaceutical composition of the present invention can be formulated as an injectable, oral preparation (tablets, capsules, oral solution), stent coating, transdermal preparation, or sustained-release formulation. The active ingredient is administered in a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be formulated as a sustained-release formulation. The pharmaceutical composition of the present invention is preferably an injectable formulation. In addition, the pharmaceutical composition of the present invention can be used in combination with other therapeutic agents. Furthermore, the pharmaceutical composition of the present invention may further comprise additional components selected from the following groups: (1) components or protein truncated polypeptides that inhibit angiotensin II and heart failure after endothelial injury; (2) components that inhibit myocardial hypertrophy; and (3) components or protein truncated polypeptides that inhibit cardiac cell fibrosis.

[0053] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated for the patient, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg / kg of animal body weight per day, preferably, 0.0001 mg-10 mg / kg of animal body weight, a satisfactory effect can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered per day, or the dose may be reduced proportionally.

[0054] The pharmaceutically acceptable carriers of the present invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration, as is well known to those skilled in the art.

[0055] (4) Treatment methods

[0056] The present invention provides a pharmaceutical composition for treating angiotensin II and heart failure after endothelial injury; myocardial cell hypertrophy; and cardiac cell fibrosis diseases using the active ingredients of the present invention or corresponding drugs.

[0057] When the active ingredient of the present invention is used for the above-mentioned purposes, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets, pills, capsules, dispersible powders, granules or suspensions (containing about 0.05-5% suspending agents), syrups (containing about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agents in an isotonic medium). For example, these pharmaceutical preparations can contain about 0.01-99%, more preferably about 0.1%-90% by weight of the active ingredient mixed with a carrier.

[0058] The two active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including but not limited to intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration. Preferred routes of administration include oral administration, intramuscular administration or intravenous administration.

[0059] From the standpoint of ease of administration, preferred pharmaceutical compositions are liquid compositions, especially injections.

[0060] This application includes at least the following beneficial technical effects:

[0061] The present invention confirms that the NUP35 gene and its protein, active truncated protein polypeptide, or its promoter can inhibit angiotensin II and heart failure after endothelial injury; inhibit myocardial cell hypertrophy; and inhibit cardiac cell fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a graph showing the results of Example 1 in which knockdown of NUP35 in cardiomyocytes aggravated angiotensin II-induced heart failure;

[0063] Figure 2 This is a graph showing the results of Example 2 showing that knockout of NUP35 in cardiomyocytes aggravates angiotensin II-induced cardiac fibrosis;

[0064] Figure 3 is a graph showing the results of RNA sequencing and RNA immunoprecipitation sequencing in Example 3;

[0065] Figure 4 This is a graph showing the results of Example 4 in which overexpression of NUP35 alleviates angiotensin II-induced heart failure;

[0066] Figure 5This is a graph showing the results of Example 5 showing that cardiomyocyte-specific overexpression of NUP35 alleviates angiotensin II-induced cardiac fibrosis (Western immunoblotting assay);

[0067] Figure 6 This is a graph showing the Western blot data in Example 6 showing that cardiomyocyte-specific overexpression of NUP35 alleviates the expression of angiotensin II-induced cardiac fibrosis-related genes Col I and Col III. DETAILED DESCRIPTION

[0068] The effects of the present application are further described in detail below with reference to the accompanying drawings and embodiments.

[0069] Example 1: Knockdown of NUP35 in cardiomyocytes aggravates angiotensin II-induced heart failure

[0070] NUP35 flox / flox Conditional knockout mice were administered Cre recombinase adeno-associated virus to knock out NUP35 protein in cardiomyocytes. Empty adeno-associated virus was administered via the tail vein as a control. One week after adeno-associated virus administration, an angiotensin II pump (1000 ng / kg / min) was implanted in the back of the mice for 2 weeks. Cardiac function was assessed by small animal ultrasound. Figure 1 As shown (the figure represents the results of 6-7 independent experiments; the data are shown as mean ± standard error. AAV-Empty represents wild-type control mice from the same littermate; AAV-Cre represents cardiomyocyte-specific NUP35 knockout mice).

[0071] Depend on Figure 1 Results showed that in the PBS-treated mice, cardiomyocyte NUP35 knockout did not affect ejection fraction (EF) or the ratio of peak diastolic mitral flow velocity E to peak diastolic mitral annular velocity e' (E / e'). However, two weeks of angiotensin II (Ang II) treatment did not affect EF but increased E / e'. Cardiomyocyte NUP35 knockout further increased E / e', suggesting that cardiomyocyte NUP35 knockout exacerbates Ang II-induced heart failure.

[0072] Example 2: Knockout of NUP35 in cardiomyocytes aggravates angiotensin II-induced cardiac fibrosis

[0073] Masson staining was performed on the heart sections of the PBS and Ang II treated control and NUP35 knockout mice. Figure 2 As shown (the figure represents the results of 6-7 independent experiments; data show the mean ± standard error; the scale bar represents 100 μm; AAV-Empty represents littermate wild-type control mice; AAV-Cre represents cardiomyocyte-specific NUP35 knockout mice).

[0074] Depend on Figure 2 The results showed that knockout of NUP35 in cardiomyocytes aggravated angiotensin II-induced cardiac fibrosis.

[0075] Example 3: RNA sequencing and RNA immunoprecipitation sequencing

[0076] RNA sequencing was performed on the heart tissues of control and NUP35 knockout mice. Figure 3 (Left) Results show that knockout of NUP35 in cardiomyocytes upregulates fibrosis and cardiac hypertrophy genes.

[0077] RNA immunoprecipitation sequencing (RIP-Seq) was performed on the NUP35 antibody, and the intersection of the RNA immunoprecipitation sequencing results and the RNA sequencing results was obtained. Figure 3 (Right) The results showed that NUP35 knockout upregulated Wif1 expression.

[0078] Example 4: Overexpression of NUP35 alleviates angiotensin II-induced heart failure

[0079] C57BL / 6 mice were injected with a cardiomyocyte-specific NUP35 overexpression adeno-associated virus and a control virus into the tail vein to overexpress NUP35 in cardiomyocytes. One week after administration of the adeno-associated virus, an angiotensin II pump (1000 ng / kg / min) was implanted in the back of the mice for two weeks. Cardiac function was assessed by small animal ultrasound. Figure 4 As shown (the figure represents the results of 6 independent experiments; the data are shown as mean ± standard error; Ctrl represents C57BL / 6 mice injected with control empty vector virus; NUP35 represents C57BL / 6 mice injected with cardiomyocyte-specific NUP35 overexpression virus).

[0080] Depend on Figure 4 The results showed that overexpression of NUP35 in cardiomyocytes alleviated angiotensin II-induced heart failure.

[0081] Example 5: Cardiomyocyte-specific overexpression of NUP35 alleviates angiotensin II-induced cardiac fibrosis (Western immunoblotting)

[0082] Western blot was performed on the heart tissues of control mice and mice with cardiomyocyte-specific overexpression of NUP35. The results are as follows Figure 5 As shown (the figure represents the results of 6 independent experiments; the data are shown as mean ± standard error; Ctrl represents C57BL / 6 mice injected with control empty vector virus; NUP35 represents C57BL / 6 mice injected with cardiomyocyte-specific NUP35 overexpression virus).

[0083] Depend on Figure 5The results showed that cardiomyocyte-specific overexpression of NUP35 alleviated the expression of cardiac fibrosis-related genes induced by angiotensin II.

[0084] Example 6: Western blot data show that cardiomyocyte-specific overexpression of NUP35 alleviates the expression of angiotensin II-induced cardiac fibrosis-related genes Col I and Col III

[0085] Immunofluorescence staining of heart sections from control mice and mice with cardiomyocyte-specific overexpression of NUP35 was performed. Figure 6 As shown (the figure represents the results of 6 independent experiments; data show the mean ± standard error; the scale bar represents 100 μm; Ctrl represents C57BL / 6 mice injected with the control empty vector virus; NUP35 represents C57BL / 6 mice injected with the cardiomyocyte-specific NUP35 overexpression virus).

[0086] Depend on Figure 6 The results showed that cardiomyocyte-specific overexpression of NUP35 slowed down the number of SMA+ myofibroblasts induced by angiotensin II.

[0087] The above are all preferred embodiments of the present application, and are not intended to limit the scope of protection of the present application. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application should be included in the scope of protection of the present application.

Claims

1. Application of NUP35 recombinant protein in the preparation of drugs for the treatment of heart failure; The NUP35 recombinant protein refers to a recombinant protein synthesized based on the amino acid sequence SEQ ID NO. 1.

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