Preparation and application of therapeutic drug for preventing and treating heart failure or myocardial ischemia-reperfusion injury

Abstract

The invention discloses preparation and application of a therapeutic drug for preventing and treating heart failure or myocardial ischemia-reperfusion injury. Research results of the invention show that the exosome secreted by kidney cells after stimulation and induction of the drug-containing serum of Zhenwu decoction has a myocardial protection effect. Therefore, the invention provides a novel candidate drug for heart failure or myocardial ischemia-reperfusion injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the preparation and application of a therapeutic drug for preventing and treating heart failure or myocardial ischemia-reperfusion injury. Background Technology

[0002] Heart failure (HF) is a complex clinical syndrome caused by various factors that lead to abnormal changes in the structure and / or function of the heart, resulting in impaired ventricular systolic and / or diastolic function. It is mainly manifested as dyspnea, fatigue, and fluid retention (pulmonary congestion, systemic congestion, and peripheral edema), and is the severe and terminal stage of most organic heart diseases.

[0003] In the field of cardiovascular disease, myocardial ischemia-reperfusion injury is a crucial clinical problem that urgently needs to be addressed. When blood flow to the heart is interrupted and then restored, the previously ischemic myocardial tissue regains its blood supply. However, this process is often accompanied by a series of complex pathophysiological reactions, such as oxidative stress, inflammatory responses, and apoptosis, leading to further damage to cardiomyocytes and even causing heart failure. Traditional treatments, such as drug therapy and interventional surgery, can alleviate the symptoms of myocardial ischemia to some extent, but their effectiveness in reducing reperfusion injury is limited.

[0004] Currently, the treatment goals for heart failure primarily focus on improving cardiac function, alleviating symptoms, and improving prognosis. Drug treatment strategies mainly include increasing cardiac output, reducing cardiac workload, and improving myocardial remodeling. Clinically, a combination of drugs such as diuretics, renin-angiotensin system (RAS) inhibitors, beta-blockers, and mineralocorticoid receptor antagonists (MRAs) is commonly used. The improvement in left ventricular ejection fraction (EF) after treatment is an important indicator for evaluating the effectiveness of heart failure treatment. Although current treatments have greatly improved patient prognosis, the morbidity and mortality of heart failure remain high, making it a major public health problem.

[0005] Therefore, it is necessary to explore new strategies for treating heart failure or myocardial ischemia-reperfusion. Summary of the Invention

[0006] This invention provides the use of kidney cell-derived exosomes in the preparation of medicaments for treating heart failure or myocardial ischemia-reperfusion injury.

[0007] Preferably, the kidney cells are renal epithelial cells.

[0008] Preferably, the kidney cells are kidney cells stimulated and induced by Zhenwu Decoction.

[0009] Preferably, the kidney cells are kidney cells stimulated and induced by serum containing Zhenwu Decoction.

[0010] More preferably, the method for preparing the Zhenwu Decoction-containing serum includes administering Zhenwu Decoction to the subject and collecting venous blood.

[0011] More preferably, the method for preparing the Zhenwu Decoction-containing serum includes administering Zhenwu Decoction to the subject for 7 days and collecting arterial blood.

[0012] Preferably, the treatment of heart failure or myocardial ischemia-reperfusion injury is achieved through the following means:

[0013] 1) Improve cardiomyocyte activity;

[0014] 2) Inhibits myocardial cell damage;

[0015] 3) Enhance left ventricular systolic function;

[0016] 4) Inhibit myocardial fibrosis.

[0017] The present invention provides a method for reversing cardiomyocyte damage in vitro, the method comprising administering Zhenwu Decoction or its drug-containing serum to cardiomyocytes.

[0018] Preferably, the method for preparing the Zhenwu Decoction-containing serum includes administering Zhenwu Decoction to the subject and collecting venous blood; the method for preparing the Zhenwu Decoction-containing serum includes administering Zhenwu Decoction to the subject for 7 days and collecting arterial blood.

[0019] This invention provides the use of a reagent that promotes miRNA-434-3p in the preparation of drugs for treating myocardial ischemia-reperfusion injury or heart failure.

[0020] In this invention, the agent that promotes miRNA-434-3p refers to any substance that can promote the activity of miRNA-434-3p, promote the expression level of miRNA-434-3p, promote the stability of the miRNA-434-3p gene, or promote the effective action time of miRNA-434-3p.

[0021] In this invention, the effects of the promoter can also be achieved by introducing an expression vector containing miRNA-434-3p into host cells. Methods well-known to those skilled in the art can be used to construct the expression vectors required for this invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells, such as resistance to kanamycin, gentamicin, hygromycin, and ampicillin. In this invention, the expression vector is any vector known in the art, such as commercially available vectors, including plasmids, granules, bacteriophages, viruses, etc. The expression vector typically also contains a promoter, origin of replication, and / or marker genes. The introduction of the expression vector into host cells can be achieved using well-known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, liposome transfection, and binding to cell membrane permeable peptides.

[0022] In this invention, the effects of promoters can also be achieved through knock-in. Knock-in refers to the targeted insertion of a transgene into the host cell genome, resulting in transgene expression and / or altered expression of the endogenous target gene (e.g., increased (including ectopic) or decreased expression), for example, by introducing an additional copy of the target gene or by operatively inserting a regulatory sequence that provides an endogenous copy of the target gene to enhance expression. Knock-in transgenes can include heterozygous knock-in or homozygous knock-in. Knock-in also encompasses conditional knock-in, wherein transgene expression and / or altered expression of the endogenous target gene can occur, for example, by exposing an animal to a substance that promotes such expression, by introducing an enzyme that promotes recombination at the targeted insertion site, or by some other method for altering the targeted insertion site.

[0023] In this invention, miRNA mimic technology can also be used to synthesize miRNA-434-3p mimics through chemical methods to mimic the high-level expression of mature miRNA-434-3p in cells, thereby enhancing the function of endogenous miRNA-434-3p.

[0024] Furthermore, the miRNA-434-3p promoter includes miRNA-434-3p itself, miRNA-434-3p mimics, or other agents that promote miRNA-434-3p.

[0025] Furthermore, the other agents that promote miRNA-434-3p include, but are not limited to, nucleic acid molecules, carbohydrates, liposomes, small molecule chemicals, antibody drugs, peptides, proteins, or reagents used in gene editing.

[0026] In this invention, the term nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As equivalents, nucleic acids also include DNA or RNA analogs derived from nucleotide analogs, and, where applicable, single-stranded (sense or antisense) and double-stranded polynucleotides. Liposomes refer to small vesicles composed of various types of lipids, phospholipids, and / or surfactants, which can be used for drug delivery to mammals. Antibodies cover complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity and fall within the scope of protection of this invention.

[0027] The present invention also provides a method for screening candidate drugs for the prevention and / or heart failure or myocardial ischemia-reperfusion injury.

[0028] Furthermore, the method includes the following steps:

[0029] (1) Contact the test substance with a system containing or expressing miRNA-434-3p;

[0030] (2) The expression level of miRNA-434-3p in the system was detected;

[0031] (3) Select substances that can promote the expression level of miRNA-434-3p as candidate drugs for the prevention and / or treatment of heart failure or myocardial ischemia-reperfusion injury.

[0032] Furthermore, the system includes (but is not limited to): cellular system, subcellular system, solution system, tissue system, organ system, or animal system.

[0033] Furthermore, the test substance includes (but is not limited to): interfering molecules, nucleic acid inhibitors, and small molecule compounds designed to target myocardial ischemia-reperfusion injury or its upstream or downstream genes.

[0034] The present invention also provides the use of miRNA-434-3p in screening candidate drugs for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

[0035] The miRNA-434-3p sequences disclosed in this invention can all be found in the miRBase database (http: / / microrna.sanger.ac.uk / ):

[0036] miRBase ID: MIMAT0001422

[0037] miRNA name: mmu-miR-434-3p (mouse-derived miR-434-3p)

[0038] Sequence: 5'-UUUGAACCAUCACUCGACUCCU-3';

[0039] miRBase ID: MIMAT0005315

[0040] miRNA name: rno-miR-434-3p mature miRNA (rat-derived miR-434-3p)

[0041] Sequence: 5'-UUUGAACCAUCACUCGACUCCU-3'

[0042] This indicates that miR-434-3p is relatively conserved across different species. Attached Figure Description

[0043] Figure 1 The results of primary cell identification of suckling mouse kidneys are shown in the figure, where (A) fluorescence image of CK-18 and AQP1; (B) fluorescence statistical image of CK-18 and AQP1;

[0044] Figure 2 Electron micrographs of exosomes are shown;

[0045] Figure 3 Display exosome particle size distribution;

[0046] Figure 4 Displays Western blot images of Calnexin, CD63, and CD9 proteins;

[0047] Figure 5 The results of ZWD-Exo improving cardiac function in mice with heart failure are shown in the figure, where (A) left ventricular echocardiography of mice in each group; (B) statistical graph of ultrasound results of mice in each group.

[0048] Figure 6 The diagram shows the effect of ZWD-Exo on left ventricular outflow tract blood flow in mice with heart failure. (A) Cross-sectional view of the left ventricular outflow tract in each group of mice; (B) Statistical graph of outflow tract blood flow results in each group of mice.

[0049] Figure 7 The graph shows the effect of ZWD-Exo on the myocardial infarction area in mice with heart failure. (A) TTC-stained heart sections in each group; (B) Statistical graph of TTC staining results.

[0050] Figure 8 The graph showing the effect of ZWD-Exo on the cardiac coefficient in mice with heart failure;

[0051] Figure 9The graph shows the effect of ZWD-Exo on CK and LDH levels in mice with heart failure. (A) Effect of ZWD-Exo on CK levels in mice with heart failure; (B) Effect of ZWD-Exo on LDH levels in mice with heart failure. Figure 10 The diagram showing the effect of ZWD-Exo on myocardial tissue in mice with heart failure;

[0052] Figure 11 The diagram shows the effect of ZWD-Exo on myocardial fibrosis in mice with heart failure. (A) Masson staining of cardiac tissue in each group; (B) Collagen volume fraction of myocardial tissue in each mouse.

[0053] Figure 12 The diagram shows the effect of ZWD-Exo on oxidative stress damage in mice with heart failure. (A) Effect of ZWD-Exo on GSH level in mice with heart failure; (B) Effect of ZWD-Exo on MDA level in mice with heart failure; (C) Effect of ZWD-Exo on SOD level in mice with heart failure.

[0054] Figure 13 The results of ZWD-Exo's protective effect on cardiomyocytes in mice with heart failure are shown in the figure. (A) TUNEL staining of myocardial tissue in each group; (B) apoptosis rate of cardiomyocytes in each group.

[0055] Figure 14 The figure shows the effect of ZWD-Exo on the expression of proteins related to the Wnt3a / β-catenin signaling pathway in mice with heart failure. (A) Western blot of Wnt3a, β-catenin, and GAPDH proteins; (B) Gray value of Wnt3a / GAPDH protein expression; (C) Gray value of β-catenin / GAPDH protein expression.

[0056] Figure 15 The figure shows the effect of ZWD-Exo on the expression of mitochondrial apoptosis-related proteins in mice with heart failure. (A) Western blot of DRP1, OPA1, Caspase9, and GAPDH proteins; (B) Gray value of DRP1 / GAPDH protein expression; (C) Gray value of OPA1 / GAPDH protein expression; (D) Gray value of Caspase9 / GAPDH protein expression.

[0057] Figure 16 The expression map of miRNA-434-3p in each group of cells is shown;

[0058] Figure 17 The effect of miRNA-434-3p on H9c2 cell viability was shown;

[0059] Figure 18The effect of miRNA-434-3p on LDH in H9c2 cells was shown;

[0060] Figure 19 The effect of miRNA-434-3p on mitochondrial membrane potential in H9c2 cells is shown in (A) JC-1 staining image; (B) effect of miRNA-434-3p on mitochondrial membrane potential in H9c2 cells.

[0061] Figure 20 The effects of miRNA-434-3p on the Wnt3a and β-catenin genes in H9c2 cells were shown, including (A) the effect of miRNA-434-3p on the Wnt3a gene in H9c2 cells; and (B) the effect of miRNA-434-3p on the β-catenin gene in H9c2 cells.

[0062] Figure 21 The figure shows the effect of miRNA-434-3p on the expression of proteins related to the Wnt3a / β-catenin signaling pathway in H9c2 cardiomyocytes. (A) Western blot of Wnt3a, β-catenin, and GAPDH proteins; (B) Gray value of Wnt3a / GAPDH protein expression; (C) Gray value of β-catenin / GAPDH protein expression. Figure 22 The figure shows the effect of miRNA-434-3p on the expression of mitochondrial apoptosis-related proteins in H9c2 cardiomyocytes. Among them, (A) Western blot of DRP1, OPA1, Caspase9, and GAPDH proteins; (B) Gray value of DRP1 / GAPDH protein expression; (C) Gray value of OPA1 / GAPDH protein expression; (D) Gray value of Caspase9 / GAPDH protein expression. Detailed Implementation

[0063] The present invention will be further illustrated below with reference to specific embodiments. However, these embodiments are merely illustrative and not intended to limit the scope of the invention. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional conditions or according to the manufacturer's recommended conditions.

[0064] The term "miRNA inhibitor" used in this article, also known as a miRNA sponge or miRNA sponge structure, refers to a chemically modified inhibitor specifically targeting miRNAs in cells. It specifically targets and knocks out individual miRNA molecules, weakening the gene silencing effect of endogenous miRNAs, increasing protein expression, and enabling loss-of-function studies. It can be used to screen miRNA target sites, miRNAs regulating the expression of specific genes, and miRNAs affecting cell development. miRNA inhibitors are chemically modified single-stranded RNAs that competitively bind to mature miRNA sequences. They are readily available, easy to use, and have short experimental cycles, making them well-suited for miRNA functional analysis studies, such as those investigating cell proliferation, apoptosis, cell differentiation, cell migration, and stem cell growth. miRNA inhibitors can be transfected into cells simply by encapsulating them with transfection reagents, eliminating the need for complex vector construction and concerns about viral protection. Transfection efficiency can be observed using a transfection control.

[0065] Example

[0066] I. Experimental Materials

[0067] - Serum containing Zhenwu Decoction (prepared by gavage administration to SD rats for 7 days)

[0068] -ICR primary kidney cells from neonatal rats (CK18 / AQP1 positive identification)

[0069] - Zhenwu decoction exosomes. Purity was determined by electron microscopy, particle size, and Western blotting (CD9 / CD63 positive, Calnexin negative).

[0070] - Mouse model of ICR in heart failure (coronary artery ligation)

[0071] Zhenwu Decoction Compound Granules - Tianjin University of Traditional Chinese Medicine Affiliated Baokang Hospital

[0072] CD9 antibody H2321, Santa Claus Corporation

[0073] CD63 antibody B2322, Santa Claus Corporation

[0074] Calenxin antibody L1321, Santa Fe Company

[0075] CK-18 antibody C48696-AF647 Signalway Antibody

[0076] II. Experimental Methods

[0077] 1. Isolation and culture of primary kidney cells

[0078] (1) Sterilize the scissors, tweezers and test tubes with high temperature steam, and immerse the petri dish in 75% alcohol for 30 minutes before use.

[0079] (2) The suckling mice were euthanized by decapitation and their bodies were sterilized by immersing them in 75% alcohol for 2 minutes. Then the abdominal cavity of the suckling mice was cut open, and both kidneys were removed and washed with PBS.

[0080] (3) Cut the kidney into pieces and put it into a 1:1 mixture of type IV collagenase and trypsin. Digest it in a 37°C water bath for 30 minutes. Shake or blow it every 5 minutes to make the tissue digest evenly. The kidney gradually becomes transparent and the tissue becomes a clot.

[0081] (4) Add twice the volume of total culture solution to stop digestion. If digestion is not complete, continue digestion in a water bath with digestion solution. After digestion, pass the tissue through a 200-mesh cell sieve and let the tissue digestion solution fall by gravity. Do not crush the tissue.

[0082] (5) Collect the filtered cell suspension and centrifuge at 1000 rpm for 10 min. Repeat the centrifugation process to purify the cells and finally obtain whole kidney primary cells. Seed the cells into cell culture flasks and culture them in an incubator. After the cells reach 80% confluence, subsequent experimental procedures will begin.

[0083] 2. Preparation of serum containing Zhenwu Decoction

[0084] SD rats were administered Zhenwu Decoction at 4 times the clinical dose (1.08 g / kg). After 7 days of gavage, blood was collected from the abdominal aorta and centrifuged at 3000 rpm to obtain serum. The drug-containing serum was then inactivated at 56°C for 30 min and filtered through a 0.22 μm microporous membrane. 500 μl of the inactivated serum was stored at -80°C for later use.

[0085] 3. Collection and identification of renal cell exosomes

[0086] (1) Preparation and purification of primary kidney cell exosomes

[0087] Primary P2 generation kidney cells were seeded into 75 cm² culture flasks and cultured in DMEM medium for 24 h, then replaced with exosome-specific medium and cultured for 48 h. The cell culture medium was then collected. Cell debris was precipitated by centrifugation at 3000 g for 30 min and transferred to ultrafiltration tubes. After concentration, the cells were centrifuged at 10000 g for 90 min, the supernatant was discarded, and the precipitate was resuspended in 200 μl PBS.

[0088] (2) Identification of exosomes from primary kidney cells

[0089] a. Morphological identification of Exo

[0090] Take 10 μL of Exo suspension and drop it onto a copper grid with a pore size of 2 nm. Let it stand at room temperature for 3-5 min, blot the liquid with filter paper, add 10 μL of 3% phosphotungstic acid solution, and negatively stain for 5 min at room temperature. Blot the negative stain with filter paper, place the copper grid under a transmission electron microscope, observe the morphology of Exo and take pictures.

[0091] b. Exosome particle size analysis using a particle size analyzer

[0092] Take 10 μL of the exosome purification solution and buffer it in 990 μL of ultrapure water. Perform particle size analysis.

[0093] c. Western blot identification of Exo expressed proteins

[0094] The selected proteins were exosomal marker CD63, negative protein Calnexin, and internal control actin.

[0095] 4. Preparation and purification of exosomes from kidney-derived cells induced by Zhenwu Decoction

[0096] P2 generation primary cells were cultured normally, and cultured in a medium containing 10% Zhenwu Decoction serum for 24 hours, then replaced with exosome-specific medium for 48 hours. The remaining operations were the same as in 3(1).

[0097] 5. miRNA throughput sequencing analysis

[0098] High-throughput sequencing technology was used to screen differentially expressed miRNAs of ZWD-Exo, and bioinformatics methods were used to screen out miRNAs associated with heart failure.

[0099] ① Extraction of total RNA from exosomes: Add 1 mL of TRIZOL reagent and 0.2 mL of chloroform to exosomes, mix well, let stand for 5 min, and then centrifuge at 10000×g for 10 min at 4℃. Transfer the supernatant to another EP tube, add 0.5 mL of isopropanol, and mix well. Incubate overnight at -20℃. Centrifuge again at 10000×g for 10 min at 4℃, discard the supernatant, add 1 mL of 75% ethanol, mix thoroughly, and continue centrifuging at 10000×g for 10 min at 4℃. Air dry at room temperature, add 30 μL of DEPC-treated ddH2O water to dissolve the RNA, and store at -80℃.

[0100] ② High-throughput sequencing: 3 μg of RNA from H2O-Exo and ZWD-Exo was used to construct libraries. Libraries were constructed using the Tru Seq SmallRNA Sample Prep Kit and quality-checked using an Agilent 2200 Tape Station. The constructed libraries were sequenced and identified using Illumina Hiseq2000, and differentially expressed miRNAs were calculated using DEGseq software based on MA-plot. Using the gene ontology (GO) database as a reference, the target genes of the differentially expressed miRNAs were statistically analyzed, and their functionality was predicted.

[0101] 6. In vivo experiments

[0102] 6.1 Modeling Method

[0103] Ninety mice were randomly divided into a normal control group (n=10) and a surgical group (n=80). The 80 mice in the surgical group were fasted for 12 hours prior to surgery. In this experiment, male ICR mice were anesthetized with 2% isoflurane gas. A small longitudinal incision of approximately 1.2 cm was made in the skin at the apex of the heart, located between the 3rd and 4th intercostal spaces on the left chest. The pectoralis major and pectoralis minor muscles were dissected layer by layer. The pleura was gently punctured with hemostats, and the heart was gently squeezed out of the chest wall by rotating the hemostats or the mouse's body. Under strong light, the left atrial appendage (LAD) was quickly located and ligated with 6 / 0 non-invasive sutures approximately 1 mm below the left atrial appendage on the line connecting the left atrial appendage and the apex of the heart. The heart was then pushed back into the thoracic cavity using the hemostats, and the cavity was quickly squeezed to expel air. The incision was sutured with 4 / 0 non-invasive sutures and disinfected. The sham surgery group underwent the same procedure but without ligation.

[0104] 6.2 Grouping and Administration

[0105] Mice with normal cardiac ejection fraction (EF) were selected for modeling. After modeling, EF values ​​were measured by echocardiography, and mice with EF values ​​greater than 50% were excluded. Subsequently, based on the EF values ​​after modeling, the mice were divided into 6 groups (n=12): sham surgery group, model group, ZWD-Exo group, PBS-Exo dosage group, and Western medicine group. The Sham group and the Model group received 0.9% saline intravenous injections via gastric vein daily for four weeks.

[0106] 6.3 Ultrasound assessment of left ventricular function and hemodynamic status

[0107] Postoperatively and 28 days after drug administration, cardiac function in mice was assessed using a Vevo 2100 small animal real-time ultrasound imaging system. The height and orientation of the MS-250 probe were adjusted. Mice were gently held and anesthetized with gas. An appropriate amount of ultrasound coupling gel was applied to the precordial region. The probe position was adjusted so that the left ventricular length, apex, outflow tract, and papillary muscle structures were visible in axial section under B-mode ultrasound. B-mode ultrasound images were then acquired. The ultrasound was then switched to M-mode to acquire M-mode ultrasound images. Each image was captured over three consecutive cardiac cycles. In M-mode ultrasound, the long axis measurement package (PLAX) was selected to measure and calculate the left ventricular ejection fraction (EF), left ventricular fractional shortening (FS), iterventricular septum thickness at systolic (IVS;s), and left ventricular end-systolic volume (LVVol;s). Using the same method, the MS-250 probe was positioned in the left ventricular outflow tract, and velocity-time integral (VTI), peak velocity (PeakVel), peak pressure (PeakGrad), mean velocity (Mean Vel), and mean pressure (Mean Girad) were measured to assess outflow tract hemodynamics. Then, using the same method, the probe was positioned in a four-chamber mitral valve, and the peak velocity (E) of early mitral valve inflow and the peak velocity (A) of late mitral valve inflow were measured. The E / A ratio was calculated to assess mitral valve blood flow.

[0108] 6.4 Blood and Heart Sample Collection

[0109] Blood sample collection: After drug administration, blood was collected from the mice via the ocular venous plexus into 1.5 mL centrifuge tubes. Centrifuged at 3500 rpm for 10 min, and the supernatant was collected and stored at -80℃ for later use.

[0110] Pathological section heart sample collection: After blood collection, the sample number is marked on the tube wall. The heart is removed, rinsed 3 times with 0.9% physiological saline, and then rinsed 2 times with 4% paraformaldehyde tissue fixative. The heart is then fixed in formalin solution and stored at room temperature for later use.

[0111] 6.5 Serum Biochemical Indicators Detection

[0112] The serum creatine kinase (CK) and glucose (GLU) of mice in each group were detected using a fully automated biochemical analyzer.

[0113] 6.6 Cardiac coefficient detection

[0114] After the drug administration was completed, the body weight of the mice was recorded. After blood collection, the mouse hearts were removed, and the weight of each heart was recorded. The mouse heart coefficient was calculated (heart coefficient = heart mass / body mass * 100%).

[0115] 6.7 Calculation of the percentage of myocardial infarction area in mice

[0116] After echocardiography, the heart tissue was removed, rinsed thoroughly in saline, and the pericardial fat was removed. The heart was then refrigerated at -80°C. Once the mouse heart had reached the appropriate firmness, it was removed and immediately sliced ​​into five uniformly thick pieces. These pieces were then added to a pre-prepared 1% TTC staining solution (0.2g TTC powder dissolved in 20mL PBS solution, stored in the dark) and incubated at 37°C for 30 minutes in the dark. After incubation, normal heart tissue appeared red, while infarcted tissue appeared white. The tissue was then fixed in 4% paraformaldehyde and photographed after 24 hours. ImageJ software was used to scan the tissue and calculate the percentage of myocardial infarction area.

[0117] 6.8 Observation of cardiac myocardial tissue pathological morphology by HE staining

[0118] 6.8.1 Preparation of Paraffin Sections from Heart Tissue

[0119] Fresh hearts were immersed in a 4% paraformaldehyde solution and fixed at 4°C for 24–48 hours. Dehydration was then performed, and the aorta was sequentially immersed in a gradient of ethanol solutions (70%, 80%, 90%, 95%, 95%, 100%, 100%) and xylene, each concentration for 1 hour. Subsequently, the tissue was immersed three times in a paraffin solution preheated to 55°C, each time for 0.5–1 hour, ensuring that paraffin completely replaced xylene for tissue embedding. The paraffin-embedded tissue was placed in a mold containing paraffin solution and embedded using an embedding machine, ensuring the cut surface was parallel to the bottom. The embedded mold was then transferred to a -10°C freezing stage to cool for 30 minutes. After the paraffin solidified, the wax block was trimmed, and sections were cut to a thickness of 4 μm. The sections were then floated on warm water in a slide facilitator to flatten them, and the tissue was retrieved using a glass slide and placed in an oven to bake. After the water dried and the paraffin melted, the sections were removed and stored at room temperature for later use.

[0120] 6.9 Hematoxylin-eosin staining (H&E staining method)

[0121] Paraffin sections of heart tissue were dewaxed and hydrated, stained with hematoxylin for 10 min, rinsed with running water, differentiated in differentiation solution for 30 s, soaked in ddH2O for 15 min, stained with eosin for 1 min, rinsed with running water, soaked in ddH2O for 3 min, dehydrated, cleared, mounted, and photographed under a microscope.

[0122] 6.10 TUNEL staining

[0123] Heart tissue paraffin sections were dewaxed and hydrated. Subsequent steps were strictly performed according to the reagent instructions. Proteinase K working solution was added to the sections, and the sections were reacted at 37°C for 15 minutes, then air-dried and mounted. Under a microscope, the nuclei of apoptotic cells showed green fluorescence.

[0124] 6.11 Masson staining

[0125] Paraffin sections of heart tissue were dewaxed and hydrated, stained overnight with potassium dichromate solution, stained with hematoxylin for 3 min, rinsed with running water, differentiated with 1% hydrochloric acid ethanol for 8 s, soaked in 0.8% ammonia water for 5 s, soaked in Ponceau S and Acid Fuchsin for 8 min, soaked in phosphomolybdic acid solution for 2 min, soaked in aniline blue solution for 5 min, and soaked in 1% glacial acetic acid for 1 min; after dehydration and mounting, they were photographed under a microscope.

[0126] 6.12 Western blot detection of protein expression

[0127] 20 mg of mouse heart tissue was weighed, and protein was extracted according to the steps of the protein extraction kit. The protein concentration was detected using the BCA method. 30 μg of protein was loaded onto each lane. After separation by SDS-PAGE using a 10% gel, the protein was transferred to a PVDF membrane and blocked with 5% skim milk powder at room temperature for 2 h. After blocking, the membrane was washed three times with TBST buffer. After washing, primary antibody reaction solution containing GAPDH (1:1000), Wnt3a, β-catenin, Caspase 9, DRP1, and OPA1 was added, and the membrane was incubated overnight at 4°C. Secondary antibody (1:10000) was added, and the membrane was incubated at room temperature for 2 h. The membrane was washed six times with TBST buffer, and ECL was used for color development. GAPDH was used as an internal control, and the gray values ​​of each electrophoretic band were measured using Image-J analysis software.

[0128] 7. Cell experiments

[0129] 7.1 Culture of rat cardiomyocytes H9c2 and construction of H / R model

[0130] H9c2 cells were cultured in high-glucose DMEM medium (basal medium) containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator (conventional incubator) until the logarithmic growth phase.

[0131] H / R model construction: H9c2 cells were cultured in serum-free sugar-free DMEM medium with 5 mmol sodium dithionite for 4 h, and then replaced with basal culture medium for routine culture for 2 h.

[0132] 7.2 Cell Transfection

[0133] Cells were seeded in 6-well plates and transfected after reaching 70%–80% confluence using RIBOBIO riboFECT CP reagent.

[0134] 7.2.1 Design and Synthesis of miRNA

[0135] The selected mRNA-434-3p was designed and synthesized.

[0136] 7.2.2 Experimental Grouping

[0137] a. Blank group;

[0138] b. Model group (H / R model);

[0139] c. miRNA overexpression group;

[0140] d. miRNA silencing group

[0141] The miRNA overexpression group should be transfected with miRNA-434-3p mimic, micrON mmu-miR-434-3pmimic, purchased from Guangzhou Ruibo Biotechnology Co., Ltd., product number: miR10001422-1-5.

[0142] The miRNA silencing group was transfected with miRNA-434-3p inhibitor, mirOFF mmu-miR-434-3pinhibitor, purchased from Guangzhou Ruibo Biotechnology Co., Ltd., product number: miR20001422-1-5.

[0143] 7.2.3 Transfection Steps

[0144] H9c2 cardiomyocytes were seeded into six-well plates using DMEM basal medium without antibiotics and divided into three groups: control group, miRNA-434-3p mimic group, and miRNA-434-3p inhibitor group. Cells were treated when the cell density reached 50-60%. Transfection reagents, 50 nmol miRNA-434-3p mimic, and 100 nmol miRNA-434-3p inhibitor were dissolved separately in DMEM basal medium. After mixing and standing for 5 min, the prepared transfection reagents were mixed with the miRNA-434-3p mimic and miRNA-434-3p inhibitor solutions, respectively, and incubated for 20 min. Finally, the two transfection complexes were added to the required wells of the plates, mixed thoroughly, and incubated at 37°C in a 5% CO2 incubator. Cells transfected for 24 h were used for subsequent experiments.

[0145] 7.3 Effects of miRNA on the proliferation of hypoxic H9c2 cells

[0146] Healthy H9c2 cells were seeded into 96-well plates at a density of 4000 cells per well, with three replicates per group. Transfection with miRNA began 24 hours after seeding. After transfection, the cells were incubated for 24 hours. The model group, miRNA overexpression group, and miRNA silencing group were then subjected to hypoxia treatment for 4 hours, followed by reoxygenation for 2 hours. The old culture medium was discarded, and a mixture of MTT and culture medium at a ratio of 1:9 was prepared, with 0.1 ml per well. This mixture was added to the 96-well plates using a multipipeline. After incubation for 4 hours, the absorbance of each well was measured at 570 nm using a microplate reader.

[0147] 7.4 Effects of miRNA on proteins in hypoxic H9c2 cells

[0148] H9c2 cells were seeded at a density of 200,000 cells per well in 6-well plates, with 2 ml of H9c2 cell culture medium added to each well. After 24 hours, grouping, transfection, and hypoxia were performed as before. After hypoxia-reoxygenation, all cells were observed under a microscope, and cell morphology was recorded by photograph. The cell supernatant and cells were collected. Dead cells and debris were removed from the supernatant, which was then stored at -20°C for later use. Cells were scraped off to prepare protein samples, following the same procedure. The prepared protein samples could be stored at -2°C initially and then analyzed by Western blotting the next day, following the same steps.

[0149] 7.5 Effects of miRNA on LDH in H9c2 cells

[0150] Collect cell supernatant and measure LDH.

[0151] 7.6 Effects of miRNA-434-3p on mitochondrial membrane potential in H9c2 cells

[0152] The JC-1 mitochondrial membrane potential detection kit was tested according to the instructions. The specific steps are as follows:

[0153] (1) Plating for drug delivery: Take H9c2 cells that have been cultured for 3-5 generations and seed them in 96-well plates at a rate of 3000 cells / well. The cell culture, transfection, and modeling methods are the same as before.

[0154] (2) Prepare JC-1 staining working solution: Take an appropriate amount of JC-1 (200x) and dilute it with 8 mL of ultrapure water for every 50 μL of JC-1 (200x). After thorough shaking and mixing, add 2 mL of JC-1 staining buffer (5X) and mix well to obtain the JC-1 staining working solution.

[0155] (3) Add working solution: Add 200 μL of JC-1 staining working solution to each well and mix thoroughly. Incubate at 37℃ for 20 minutes.

[0156] (4) Prepare buffer solution: During incubation, add 16 mL of ultrapure water to every 4 mL of JC-1 staining buffer to prepare JC-1 staining buffer (1×).

[0157] (5) Washing: After incubation, discard the supernatant and wash twice with 200 μL JC-1 staining buffer (1×) per well.

[0158] (6) Observation and detection: Add 100 μL of cell culture medium, observe under a fluorescence microscope, and take pictures to analyze the mitochondrial membrane potential level of H9c2 cells in each group.

[0159] 7.7 Effects of miRNA-434-3p on Wnt3a and β-catenin genes in H9c2 cells

[0160] RNA extraction from H9c2 cells and RT-PCR detection of Wnt3a and β-catenin gene expression. III. Experimental Results

[0161] ZWD-Exo regulates the Wnt3a / β-catenin pathway via miRNA-434-3p, maintaining mitochondrial fission-fusion balance (DRP1↓ / OPA1↑) and reducing cardiomyocyte apoptosis.

[0162] like Figure 1 As shown, renal tubular epithelial cells were identified using the epithelial-specific marker CK18 and the proximal tubular cell-specific marker AQP1. After staining the cells with DAPI, the cell nuclei appeared as blue, pebble-like oval outlines under a fluorescence microscope. Approximately 95% of the cells showed positive CK18 staining in the cytoplasm, confirming that the cells were epithelial cells; approximately 90% of the cells expressed AQP1 in the cytoplasm, confirming that the cells were proximal tubular epithelial cells.

[0163] like Figure 2 As shown, transmission electron microscopy revealed that the diameters of primary renal cell exosomes were roughly distributed in the range of 50 to 150 nanometers, and their overall shape tended to be uniform, exhibiting a typical elliptical morphology.

[0164] like Figure 3 As shown, the average size of H2O-Exo exosomes was 115.31 nm and the average size of ZWD-Exo exosomes was 156.84 nm, as determined by instrumental analysis.

[0165] like Figure 4 The experimental results showed that, compared with the proteins in the lysate of primary renal cells, the exosome surface markers CD9 and CD63 extracted in this experiment were positively expressed, while the calcium-binding protein Calnexin on the endoplasmic reticulum membrane was negatively expressed.

[0166] The structure and function of the mouse heart were assessed after ultrasound examination of the long-axis section of the left ventricle. Figure 5 As shown, compared with the Sham group, the EF, FS, and IVS;s of the Model group were significantly decreased (P<0.01), while LVvol;s and LVID;s were significantly increased (P<0.01). Compared with the Model group, H2O-Exo (10 μg / ml) intervention increased the EF value from 36.39% to 65.51% (P<0.001) and the FS value from 14.66% to 30.92% (P<0.001), while LVvol;s decreased from 43.64% to 21.82% (P<0.01) and LVID;s decreased from 24.82% to 18.64% (P<0.05). After administration of ZWD-Exo (10 μg / ml), the EF value increased from 36.39% to 79.04% (P<0.001), the FS value increased from 14.66% to 41.92% (P<0.001), and the LVS value increased from 1.5% to 1.818% (P<0.05). At the same time, the LV vol value decreased from 43.64% to 18.18% (P<0.01), and the LVID value decreased from 24.82% to 18.73% (P<0.01). DAPA administration increased EF from 36.39% to 79.76% (P<0.001), FS from 14.66% to 46.66% (P<0.001), and LVPW;s from 18.91% to 20.18% (P<0.05), while LVvol;s decreased from 43.64% to 9.091% (P<0.001).

[0167] High-resolution small animal color ultrasound was used to detect parameters such as VTI, Mean Grad, Mean Vel, Peak Grad, and Peak Vel. The effect of ZWD-Exo on left ventricular outflow tract blood flow in heart failure mice was observed. Figure 6Compared with the Sham group, the VTI, Mean Grad, Mean Vel, Peak Grad, and Peak Vel were all significantly reduced in the Model group mice (P<0.001). Compared with the Model group, after ZWD-Exo (10 μg / ml) administration, VTI increased from 14.06% to 21.13% (P<0.001), Mean Grad increased from 39.18% to 71.91% (P<0.001), Mean Vel increased from 273.5% to 363.7% (P<0.001), and Peak Grad increased from 83.82% to 173.7% (P<0.001). DAPA intervention increased VTI from 14.06% to 21.63% (P<0.001), Mean Grad from 39.18% to 71.45% (P<0.001), and MeanVel from 273.5% to 366.2% (P<0.001).

[0168] like Figure 7 As shown, compared with the Sham group, the myocardial infarction area in the Model group mice increased by 273% (P<0.001). However, compared with the Model group, the myocardial infarction area in mice decreased from 61.88% to 19.13% after ZWD-Exo (10 μg / ml) administration (P<0.001); and the myocardial infarction area in mice decreased from 61.88% to 18.76% after DAPA administration (P<0.001).

[0169] like Figure 8 As shown, compared with the Sham group, the heart coefficient of the Model group mice was significantly higher than that of the Sham group by 15.3% (P<0.05), while the heart coefficient of the ZWD-Exo (10μg / ml) group mice was significantly lower by 11.92% (P<0.05).

[0170] like Figure 9 As shown, compared with the Sham group, the CK and LDH levels in the Model group mice were significantly higher than those in the Sham group by 64.07% (P<0.001) and 68.8% (P<0.01), respectively. The ZWD-Exo (10 μg / ml) group mice showed significantly reduced CK and LDH levels by 61.68% (P<0.001) and 60.14% (P<0.05), respectively. The DAPA group showed a significantly reduced CK level by 49.7% (P<0.01).

[0171] After observation Figure 10HE staining results clearly showed no obvious pathological abnormalities in the cardiomyocytes of the Sham group mice; the cardiomyocytes were neatly arranged and clearly structured. In contrast, the cardiomyocytes of the Model group mice exhibited significant pathological changes, with disordered arrangement, widened gaps, shrunken cell morphology, and extensive infiltration of inflammatory cells. In the experimental groups treated with ZWD-Exo (10 μg / ml) and DAPA, compared with the Model group, the rupture of mouse cardiomyocytes was significantly reduced, the arrangement was more neat, the morphology and structure were clearer, and the infiltration of inflammatory cells was relatively less. This indicates that ZWD-Exo significantly improves the damage to mouse cardiomyocytes, can slow down the infiltration of inflammatory cells, and effectively alleviate the damage to cardiomyocytes in mice with heart failure.

[0172] like Figure 11 As shown, compared with the Sham group, the Model group mice exhibited disordered myocardial fiber arrangement with widened gaps, increased collagen, and myocardial fiber rupture, with a significantly increased collagen volume fraction (CVF) (P<0.001). In contrast, after drug treatment, the ZWD-Exo and DAPA groups showed a more orderly arrangement of myocardial fibers, with only a small amount of collagen observed, and a significantly reduced collagen volume fraction (P<0.001).

[0173] like Figure 12 As shown, compared with the Sham group, the Model group showed a significant decrease in SOD and GSH levels of 37.32% (P<0.05) and 51.08% (P<0.05), respectively, and a significant increase in MDA levels of 198.9% (P<0.05), indicating that the Model group showed significant differences in all three indicators. ZWD-Exo (10 μg / ml) administration significantly increased SOD and GSH levels by 55.75% (P<0.05) and 158.9% (P<0.05), respectively, and decreased MDA levels by 34.66% (P<0.05). Simultaneously, DAPA significantly decreased MDA levels by 42.08% (P<0.05).

[0174] like Figure 13 As shown, the apoptosis rate of cardiomyocytes in the Model group mice was 60.09%, which was significantly higher than that in the Sham group by 193.9% (P<0.01). In the ZWD-Exo (10 μg / ml) group, cardiomyocyte apoptosis was significantly reduced by 56.93% after administration (P<0.01). Meanwhile, the apoptosis rate of cardiomyocytes in the DAPA-treated group was significantly reduced by 64.19% (P<0.01).

[0175] like Figure 14As shown in (B), compared with the Sham group, the expression level of Wnt3a protein in the Model group mice was significantly increased by 41.26% (P<0.05). After ZWD-Exo administration, it was significantly reduced by 24.71% (P<0.05). Figure 14 As shown in (C), compared with the Sham group, the expression of β-catenin protein in the Model group was significantly increased by 31.39% (P<0.05). ZWD-Exo administration had a significant effect on the expression level of β-catenin protein, showing a significant decrease of 30.55% (P<0.05). Similarly, DAPA administration also significantly reduced β-catenin protein expression by 24.55% (P<0.05). Protein results are as follows. Figure 14 As shown in (A).

[0176] Protein results as follows Figure 15 As shown in (B, D), compared with the Sham group, the expression of DRP1 and Caspase9 proteins in the Model group mice was significantly increased by 52.05% (P<0.05) and 33.47% (P<0.05), respectively. After ZWD-Exo administration, the expression of DRP1 and Caspase9 proteins was significantly decreased by 15.43% (P<0.05) and 26.23% (P<0.05), respectively. Simultaneously, after DAPA administration, the expression of DRP1 and Caspase9 proteins was also significantly decreased by 43.29% (P<0.001) and 21.14% (P<0.05), respectively. Compared with the Sham group, the expression of OPA1 protein in the Model group was significantly decreased by 40.33% (P<0.05). After ZWD-Exo administration, the expression of OPA1 protein was significantly increased by 70.12% (P<0.001). Simultaneously, after DAPA administration, the expression of OPA1 protein was also significantly increased by 71.74% (P<0.05). OPA1 protein expression levels are shown in the table. Figure 15 (A, C)

[0177] like Figure 16 As shown, the expression of miRNA-434-3p in H9c2 cells after transfection with miRNA-434-3p mimic and miRNA-434-3p inhibitor was significantly increased in the miRNA-434-3p mimic group compared with the con group (P<0.001), indicating that we successfully transfected miRNA-434-3p mimic and miRNA-434-3p inhibitor into H9c2 cells, which is sufficient for further experiments.

[0178] like Figure 17As shown, compared with the control group, the cell viability of the H / R group decreased significantly by 45.02% (p<0.001), indicating that the H / R model was successfully constructed. The cell viability of the miRNA-434-3p mimic+H / R group was significantly increased by 24.85% compared with the H / R group (###P<0.001), indicating that miRNA-434-3p can improve the cell viability of H / R-damaged cardiomyocytes. The cell viability of the miRNA-434-3p inhibitor+H / R group showed no significant change compared with the H / R group.

[0179] like Figure 18 As shown, compared with the Control group, the LDH level in the H / R group was significantly higher than that in the Control group by 81.5% (P<0.001). After cell transformation, compared with the H / R group, the LDH level in the miRNA-434-3p mimic+H / R group was significantly reduced by 72.72% (P<0.001). There was no significant change in cell viability between the miRNA-434-3p inhibitor+H / R group and the H / R group.

[0180] like Figure 19 As shown, compared with the control group, the green / red fluorescence value of the mitochondrial membrane potential in the H / R group decreased significantly by 58.96% (P<0.05), indicating that the mitochondrial membrane potential in the H / R model group was lower. In contrast, the green / red fluorescence value of the mitochondrial membrane potential in the miRNA-434-3p inhibitor+H / R group increased significantly by 56.73% compared with the H / R group (P<0.01). There was no significant change in cell viability between the miRNA-434-3p inhibitor+H / R group and the H / R group.

[0181] like Figure 20 As shown in (A), the expression of the Wnt3a gene was significantly reduced by 40.51% in the miRNA-434-3p mimic+H / R group compared to the H / R group (p<0.05). There was no significant change in cell viability between the miRNA-434-3p inhibitor+H / R group and the H / R group. Figure 20 As shown in (B), the expression of β-catenin gene in the miRNA-434-3p mimic+H / R group was significantly reduced by 63.19% compared with the H / R group (p<0.05). There was no significant change in cell viability between the miRNA-434-3pinhibitor+H / R group and the H / R group.

[0182] like Figure 21As shown in (B), compared with the Control group, the expression level of Wnt3a protein in the H / R group was significantly increased by 27.12% (P<0.01). After transfection with miRNA-434-3p mimic, compared with the H / R group, the expression level of Wnt3a protein in the miRNA-434-3p mimic group was significantly decreased by 27.06% (P<0.01); the miRNA-434-3p mimic+H / R group showed a significant decrease of 21.13% (P<0.01). There was no significant change in cell viability between the miRNA-434-3pinhibitor+H / R group and the H / R group. Figure 21 As shown in (C), compared with the Control group, the expression of β-catenin protein in the H / R group was significantly increased by 38.42% (P<0.05). After transfection with miRNA-434-3p mimic, compared with the H / R group, the expression level of β-catenin protein in the miRNA-434-3p mimic group was significantly decreased by 31.21% (P<0.05); the miRNA-434-3p mimic+H / R group showed a significant decrease of 30.77% (P<0.01). There was no significant change in cell viability between the miRNA-434-3p inhibitor+H / R group and the H / R group. Protein results are as follows. Figure 21 As shown in (A).

[0183] Protein results as follows Figure 22As shown in (B, D), compared with the Control group, the expression of DRP1 and Caspase9 proteins in the Control+H / R group was significantly increased by 30.06% (P<0.05) and 33.79% (P<0.01), respectively. After transfection with miRNA-434-3p mimic, compared with the Control+H / R group, the expression of DRP1 and Caspase9 proteins in the miRNA-434-3p mimic group was significantly decreased by 25.67% (P<0.05) and 24.24% (P<0.05), respectively. Simultaneously, the expression of DRP1 and Caspase9 proteins in the miRNA-434-3p mimic+H / R group was also significantly decreased by 32.91% (P<0.05) and 26.24% (P<0.01), respectively. Compared with the Control group, the expression of OPA1 protein in the Control+H / R group was significantly decreased by 49.71% (P<0.01). Cell viability in the miRNA-434-3p inhibitor+H / R group was not significantly different from that in the H / R group. After transfection with miRNA-434-3p mimic, OPA1 protein expression in the miRNA-434-3p mimic group was significantly increased by 85.15% compared to the Control+H / R group (P<0.01). Simultaneously, OPA1 protein expression in the miRNA-434-3p mimic+H / R group was also significantly increased by 60.65% (P<0.01). Cell viability in the miRNA-434-3p inhibitor+H / R group was not significantly different from that in the H / R group. OPA1 protein expression levels are shown in […]. Figure 22 (A, C)

[0184] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of kidney cell-derived exosomes in the preparation of drugs for treating heart failure or myocardial ischemia-reperfusion injury.

2. The application according to claim 1, characterized in that, The kidney cells mentioned are renal epithelial cells.

3. The application according to claim 2, characterized in that, The kidney cells mentioned are kidney cells induced by Zhenwu Decoction stimulation.

4. The application according to claim 3, characterized in that, The kidney cells mentioned are kidney cells that have been stimulated and induced by serum containing Zhenwu Decoction.

5. The application according to claim 4, characterized in that, The method for preparing the serum containing Zhenwu Decoction includes administering Zhenwu Decoction to the subject and collecting venous blood.

6. The application according to claim 5, characterized in that, The method for preparing the serum containing Zhenwu Decoction includes administering Zhenwu Decoction to the subject for 7 days and collecting arterial blood.

7. The application according to claim 1, characterized in that, The treatment of heart failure or myocardial ischemia-reperfusion injury is achieved through the following methods: 1) Improve cardiomyocyte activity; 2) Inhibits myocardial cell damage; 3) Enhance left ventricular systolic function; 4) Inhibit myocardial fibrosis.

8. A method for reversing cardiomyocyte damage in vitro, characterized in that, The method includes administering Zhenwu Decoction or its drug-containing serum to cardiomyocytes.

9. The method according to claim 8, characterized in that, The method for preparing the serum containing Zhenwu Decoction includes administering Zhenwu Decoction to the subject and collecting venous blood; the method for preparing the serum containing Zhenwu Decoction includes administering Zhenwu Decoction to the subject for 7 days and collecting arterial blood.

10. The use of a reagent that promotes miRNA-434-3p in the preparation of a drug for treating heart failure or myocardial ischemia-reperfusion injury, preferably, the reagent that promotes miRNA-434-3p is miRNA-434-3p or a mimic thereof.

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