Use of lrrc55 inhibitors for the preparation of a medicament for the prevention or treatment of myocardial ischemia-reperfusion injury

Abstract

The application provides application of Lrrc55 inhibitors in preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury, and belongs to the technical field of biological medicine.The application provides application of Lrrc55 inhibitors in preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury.The application discloses, for the first time, that Lrrc55 participates in the pathological process of myocardial ischemia-reperfusion injury through in-vivo functional intervention, and proves that targeted down-regulation of Lrrc55 expression can produce a protective effect on myocardial injury.The experimental results show that after Lrrc55 is knocked down or inhibited, the apoptosis and necrosis levels of myocardial cells after myocardial ischemia-reperfusion can be effectively reduced, myocardial interstitial collagen deposition can be reduced, the process of cardiac fibrosis can be relieved, and the left ventricular systolic function can be improved on the whole level, so that the adverse myocardial remodeling can be inhibited.The application provides a new molecular target and gene therapy strategy for prevention and treatment of myocardial ischemia-reperfusion injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Lrrc55 inhibitors in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury. Background Technology

[0002] Ischemic-reperfusion injury (IRI) refers to cellular damage and dysfunction of the myocardium following the restoration of blood supply after ischemia, and is commonly seen in reperfusion therapy after acute myocardial infarction. IRI is a significant prognostic factor after reperfusion therapy, and its pathogenesis involves multiple mechanisms, including enhanced oxidative stress, calcium homeostasis disturbance, impaired mitochondrial function, and activated inflammatory responses. In the early stages of reperfusion, reactive oxygen species (ROS) accumulate rapidly, inducing lipid peroxidation and protein oxidative modification. Simultaneously, intracellular calcium overload easily triggers abnormal opening of the mitochondrial permeability transition pore, further amplifying cellular damage signals. In addition, inflammatory cell infiltration and inflammasome activation are also believed to be involved in the progression of IRI.

[0003] Lrrc55 (leucine-rich repeat-containing protein 55) has a typical leucine repeat sequence (LxxLxLxxN) structure. This domain typically consists of 19-29 amino acids and plays an important role in mediating protein-protein interactions. Current functional studies of Lrrc55 are concentrated in fields such as neuroscience, and its expression patterns and functional roles in the cardiovascular system remain unclear. To date, no literature or patent reports have revealed changes in Lrrc55 expression in myocardial ischemia-reperfusion injury or its association with the injury process, nor have any technical solutions for using Lrrc55 as a target for cardiovascular disease intervention been found. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of Lrrc55 inhibitors in the preparation of drugs for the prevention and treatment of myocardial ischemia-reperfusion injury, to clarify the value of Lrrc55 as a therapeutic target for myocardial ischemia-reperfusion injury, to solve the problem of the lack of myocardial protective interventions targeting this molecular target in the prior art, and to provide a new direction for drug development for the treatment of related clinical diseases.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of Lrrc55 inhibitors in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury.

[0006] The present invention also provides the application of Lrrc55 inhibitors in the preparation of drugs for the prevention and treatment of any of the following diseases: (1) cardiomyocyte apoptosis; (2) cardiomyocyte necrosis; (3) cardiac fibrosis; (4) decreased cardiac function; (5) pathological ventricular remodeling after myocardial ischemia-reperfusion; (6) heart failure.

[0007] Preferably, the Lrrc55 inhibitor includes a nucleic acid molecule or reagent capable of downregulating the expression level of the Lrrc55 gene; or includes a reagent capable of inhibiting the activity of the Lrrc55 protein.

[0008] Preferably, the nucleic acid molecule includes at least one of small interfering RNA, short hairpin RNA, microRNA, and antisense oligonucleotide that targets the Lrrc55 gene.

[0009] Preferably, the short hairpin RNA is transcribed in a host cell from a coding sequence, the coding sequence comprising the positive-strand nucleotide sequence shown in SEQ ID NO.1 and the anti-strand nucleotide sequence shown in SEQ ID NO.2.

[0010] Preferably, the reagent capable of downregulating the expression level of the Lrrc55 gene includes a recombinant adeno-associated virus vector carrying an Lrrc55-shRNA coding sequence; the Lrrc55-shRNA coding sequence includes the positive-strand nucleotide sequence shown in SEQ ID NO.1 and the anti-strand nucleotide sequence shown in SEQ ID NO.2; the serotype of the recombinant adeno-associated virus vector is AAV9.

[0011] The present invention also provides a drug for preventing and / or treating myocardial ischemia-reperfusion injury, said drug being able to inhibit the expression of the Lrrc55 gene or Lrrc55 protein.

[0012] Preferably, the active ingredient of the drug includes short hairpin RNA (shRNA), which is transcribed in a host cell from a coding sequence, the coding sequence including the positive-strand nucleotide sequence shown in SEQ ID NO.1 and the anti-strand nucleotide sequence shown in SEQ ID NO.2.

[0013] The present invention also provides a method for screening drugs for the prevention and treatment of myocardial ischemia-reperfusion injury, wherein the effect of the drug to be tested on the expression level of Lrrc55 gene or its protein is detected. If the drug to be tested can reduce the expression level of Lrrc55 gene or its protein, it indicates that the drug to be tested has a potential effect on the prevention and treatment of myocardial ischemia-reperfusion injury.

[0014] The beneficial effects of this invention are: This invention, through in vivo functional intervention, reveals for the first time the involvement of Lrrc55 in the pathological process of myocardial ischemia-reperfusion injury and confirms that targeted downregulation of Lrrc55 expression can protect against myocardial injury. Experimental results show that knocking down or inhibiting Lrrc55 can effectively reduce the level of cardiomyocyte apoptosis and necrosis after myocardial ischemia-reperfusion, alleviate myocardial interstitial collagen deposition, slow the progression of cardiac fibrosis, and improve left ventricular systolic function at the overall level, thereby inhibiting adverse myocardial remodeling.

[0015] This invention constructs a recombinant adeno-associated virus vector pENN-AAV-U6-sh-Lrrc55 containing a short hairpin RNA coding sequence targeting Lrrc55. After packaging with AAV9 virus, the vector is injected via tail vein into mouse cardiomyocytes, where it transcribes specific shRNA to achieve highly efficient knockdown of endogenous Lrrc55. Experiments have demonstrated that this gene intervention significantly reduces cardiomyocyte apoptosis and necrosis, decreases the degree of cardiac fibrosis, improves cardiac function indicators, and effectively inhibits the progression of pathological ventricular remodeling. Based on this, this invention not only reveals the key pathogenic role of Lrrc55 in myocardial ischemia-reperfusion injury but also provides a novel molecular target and gene therapy pathway for the prevention and treatment of this type of injury and related heart failure, possessing significant clinical application value and translational potential. Attached Figure Description

[0016] Figure 1 This is a skeletal carrier atlas.

[0017] Figure 2 This is a partial result obtained from sequencing the sh-Lrrc55 coding sequence on a recombinant adeno-associated virus vector.

[0018] Figure 3 The figure shows the results of RT-qPCR comparison of the relative expression levels of the Lrrc55 gene between AAV9-scramble and AAV9-sh-Lrrc55 in mouse models of sham surgery and acute myocardial ischemia-reperfusion injury (AIRI). ns indicates p > 0.05. This means p < 0.001.

[0019] Figure 4 Figure 1 shows the experimental results of TUNEL and α-actinin co-staining to assess the effect of Lrrc55 knockdown on cardiomyocyte apoptosis in mice with acute myocardial ischemia-reperfusion injury. A represents a representative image of mouse myocardial tissue co-stained with TUNEL and α-actinin, and B shows the statistical analysis results of cardiomyocyte apoptosis rates in each experimental group. ns indicates p > 0.05. This means p < 0.001.

[0020] Figure 5 The effect of knocking down Lrrc55 on serum lactate dehydrogenase (LDH) activity in mice with acute myocardial ischemia-reperfusion injury is shown in the figure. ns indicates p > 0.05. This indicates that p < 0.05. This means p < 0.01.

[0021] Figure 6 The figure shows the results of RT-qPCR comparison of the relative Lrrc55 gene expression levels of AAV9-scramble and AAV9-sh-Lrrc55 in sham-operated and chronic myocardial ischemia-reperfusion injury (IRI) mouse models. ns indicates p > 0.05. This indicates that p < 0.01. This means p < 0.001.

[0022] Figure 7 Figure 1 shows the echocardiographic assessment of the effect of Lrrc55 knockdown on cardiac function in mice with chronic myocardial ischemia-reperfusion injury. A represents a representative M-mode echocardiographic image of the mouse heart; B shows the statistical results of left ventricular ejection fraction (EF); and C shows the statistical results of left ventricular fractional shortening (FS). ns indicates p > 0.05. This indicates that p < 0.05. This means p < 0.001.

[0023] Figure 8 The image shows the effects of Lrrc55 knockdown on cardiac fibrosis in mice with chronic myocardial ischemia-reperfusion injury, with A representing a representative image of mouse cardiac tissue stained with marshond staining, and B showing the statistical analysis results of the percentage of cardiac fibrosis in each experimental group. ns indicates p > 0.05. This means p < 0.001. Detailed Implementation

[0024] This invention provides the application of Lrrc55 inhibitors in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury.

[0025] In this invention, "prevention and / or treatment" refers to prevention and / or treatment. In this invention, the myocardial ischemia-reperfusion injury preferably includes at least one of the following pathological changes: increased cardiomyocyte apoptosis; cardiomyocyte necrosis; cardiac fibrosis; pathological ventricular remodeling; decreased cardiac function; and heart failure. In this invention, the decreased cardiac function caused by myocardial ischemia-reperfusion injury is mediated by multiple pathological processes, including cardiomyocyte apoptosis, cardiomyocyte necrosis, and cardiac fibrosis. During myocardial ischemia-reperfusion, cardiomyocytes undergo necrosis due to ischemia and hypoxia. Simultaneously, the apoptosis signaling pathway is activated, triggering cardiomyocyte apoptosis. After the loss of a large number of cardiomyocytes, the cardiac tissue initiates a fibrotic repair response. Excessive cardiac fibrosis leads to increased myocardial stiffness and impaired ventricular diastolic and systolic function. The synergistic effect of these three factors ultimately results in a significant decrease in cardiac function. Knocking down or inhibiting Lrrc55 can intervene at multiple pathological stages mentioned above, thereby improving the effect of decreased cardiac function.

[0026] This invention also provides the application of Lrrc55 inhibitors in the preparation of drugs for the prevention and treatment of any of the following diseases: (1) cardiomyocyte apoptosis; (2) cardiomyocyte necrosis; (3) cardiac fibrosis; (4) decreased cardiac function; (5) pathological ventricular remodeling after myocardial ischemia-reperfusion; (6) heart failure. Cardiomyocyte apoptosis, necrosis, and excessive fibrosis after myocardial ischemia-reperfusion can induce pathological ventricular remodeling. Knocking down or inhibiting Lrrc55 can block the initiation and progression of this remodeling and prevent it from transforming into the decompensated stage, providing a new direction for drug preparation for the prevention and treatment of pathological ventricular remodeling after myocardial ischemia-reperfusion.

[0027] In this invention, the Lrrc55 inhibitor preferably comprises a nucleic acid molecule or reagent capable of downregulating the expression level of the Lrrc55 gene; or comprises a reagent capable of inhibiting the activity of the Lrrc55 protein. The nucleic acid molecule preferably comprises at least one of small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), and antisense oligonucleotide (ASO) targeting the Lrrc55 gene. The short hairpin RNA is transcribed in a host cell from a coding sequence comprising the positive-strand nucleotide sequence of SEQ ID NO.1: 5'-GATCGCGTGCTGGATTTGCGAAACACTCGAGTGTTTCGCAAATCCAGCACGCTTTTTG-3' and the anti-strand nucleotide sequence of SEQ ID NO.2: 5'-AATTCAAAAAGCGTGCTGGATTTGCGAAACACTCGAGTGTTTCGCAAATCCAGCACGC-3'. Using the coding sequence of the short hairpin RNA formed by annealing the positive strand nucleotide sequence shown in SEQ ID NO.1 and the anti strand nucleotide sequence shown in SEQ ID NO.2 provided by this invention, short hairpin RNA (stem-loop structure) is transcribed in host cells, which is the key to its RNA interference function. It can efficiently mediate the knockdown of Lrrc55 gene expression and can be stably expressed in cardiomyocytes to achieve continuous knockdown of Lrrc55.

[0028] In this invention, the nucleic acid molecules preferably also include small molecule compounds, natural products, and their derivatives capable of inhibiting the transcription or translation of the Lrrc55 gene; the reagents capable of inhibiting the activity of the Lrrc55 protein preferably include monoclonal antibodies, polyclonal antibodies, antigen-binding fragments, etc., capable of specifically binding to the Lrrc55 protein and inhibiting its activity. In this invention, the reagents preferably also include recombinant vectors expressing the above-mentioned nucleic acid substances or antibodies, recombinant viruses (such as adenovirus, lentivirus, adeno-associated virus, etc.), recombinant engineered bacteria, etc.

[0029] In this invention, the reagent is preferably delivered to myocardial tissue via a recombinant viral vector, preferably an adeno-associated virus (AAV) vector, and preferably the AAV9 serotype, to improve the transduction efficiency to myocardial tissue.

[0030] In a preferred embodiment of the present invention, the reagent for knocking down or inhibiting Lrrc55 is preferably a recombinant adeno-associated virus vector loaded with the coding sequence of Lrrc55-shRNA. The coding sequence of Lrrc55-shRNA is inserted into the adeno-associated virus vector (preferably pENN-AAV-U6-shRLuc-eGFP, as shown in the diagram). Figure 1A recombinant adeno-associated virus (AAV) vector was constructed using a multiple cloning site (as shown). This vector can be used as a gene delivery tool to efficiently deliver the Lrrc55-shRNA coding sequence into cardiomyocytes. Leveraging the host cell invasion characteristics of AAV, the Lrrc55-shRNA coding sequence is transcribed and expressed in cardiomyocytes, thereby knocking down Lrrc55. Compared to naked shRNA coding sequences, the recombinant AAV vector significantly improves the cardiomyocyte delivery efficiency and expression stability of the shRNA coding sequence. More preferably, the serotype of the recombinant AAV vector is AAV9. The AAV9 serotype has natural cardiomyocyte targeting, can efficiently cross the vascular endothelial barrier and cardiomyocyte membrane, achieving specific infection of cardiomyocytes. Furthermore, AAV9 is non-pathogenic, has low immunogenicity, and has a long expression duration in vivo, mediating the long-term stable expression of the Lrrc55-shRNA coding sequence in cardiomyocytes, continuously knocking down the Lrrc55 gene. Simultaneously, it can reduce the immune response induced by the vector in vivo, improving the safety and efficacy of clinical applications, making it a preferred serotype for myocardial tissue gene therapy. The present invention does not impose any particular limitation on the construction method of the recombinant adeno-associated virus vector. A positive recombinant expression vector containing the sh-Lrrc55 coding sequence can be obtained by using conventional gene cloning methods in the art.

[0031] In this invention, the myocardial ischemia-reperfusion injury preferably includes acute myocardial ischemia-reperfusion injury or chronic myocardial ischemia-reperfusion injury.

[0032] The present invention also provides a drug for preventing and / or treating myocardial ischemia-reperfusion injury, said drug being able to inhibit the expression of the Lrrc55 gene or Lrrc55 protein.

[0033] In this invention, the medicament preferably comprises an active ingredient and at least one pharmaceutically acceptable excipient. The active ingredient improves cardiac function and inhibits the progression of myocardial ischemia-reperfusion injury by regulating Lrrc55, while the excipient provides the active ingredient with physicochemical stability, delivery assistance, and dosage form compatibility support.

[0034] In this invention, the active ingredient of the drug preferably includes short hairpin RNA, which is transcribed from a coding sequence in a host cell. The coding sequence includes the positive-strand nucleotide sequence shown in SEQ ID NO.1 and the anti-strand nucleotide sequence shown in SEQ ID NO.2. The pharmaceutically acceptable excipients preferably include any one or more combinations of buffers, encapsulating agents, fillers, transdermal absorption agents, absorption enhancers, diluents, binders, disintegrants, lubricants, flow aids, surfactants, plasticizers, stabilizers, lyophilization preservatives, and adsorbents. Those skilled in the art can select appropriate excipients and conventional dosages according to the drug dosage form requirements.

[0035] This invention does not impose any particular limitations on the preparation of reagents for knocking down or inhibiting Lrrc55, the packaging of recombinant adeno-associated virus vectors, and the preparation process of pharmaceutical compositions; these can be accomplished using conventional genetic engineering, viral packaging, and pharmaceutical formulation techniques in the field.

[0036] The present invention also provides a method for screening drugs for the prevention and treatment of myocardial ischemia-reperfusion injury, wherein the effect of the drug to be tested on the expression level of Lrrc55 gene or its protein is detected. If the drug to be tested can reduce the expression level of Lrrc55 gene or its protein, it indicates that the drug to be tested has a potential effect on the prevention and treatment of myocardial ischemia-reperfusion injury.

[0037] This invention does not specifically limit the method for detecting the effect of the drug to be tested on the expression level of the Lrrc55 gene or its protein; any conventional method in the art for detecting the expression level of a gene or its protein can be used.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the following embodiments are all conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Example 1 1. Construction and packaging of AAV9-sh-Lrrc55 adeno-associated virus vector (1) The complete CDS sequence of the Lrrc55 gene was obtained by searching the NCBI database. A DNA fragment encoding a short hairpin shRNA was designed and synthesized targeting the Lrrc55 gene-specific target site sequence. The fragment has sticky ends that match the vector at both ends. The positive strand nucleotide sequence is shown in SEQ ID NO.1: 5'-GATCGCGTGCTGGATTTGCGAAACACTCGAGTGTTTCGCAAATCCAGCACGCTTTTTG-3'; the reverse strand nucleotide sequence is shown in SEQ ID NO.2: 5'-AATTCAAAAAGCGTGCTGGATTTGCGAAACACTCGAGTGTTTCGCAAATCCAGCACGC-3'. The synthesized DNA fragments were then annealed: The reaction solution was prepared according to the system, with 5 μL of a 1 μg / μL forward DNA fragment, 5 μL of a 1 μg / μL reverse DNA fragment, and 5 μL of 10×NEB buffer 2. Sterile double-distilled water (ddH2O) was added to bring the volume to 35 μL. The reaction system was incubated at 95℃ for 5 min, then naturally cooled to room temperature to complete the annealing, forming a double-stranded DNA fragment with sticky ends. Using the appropriate restriction endonucleases BamHI and EcoRI, the shRNA expression vector pENN-AAV-U6-shRLuc-eGFP (image shown) was expressed. Figure 1 The vector was digested with enzymes (as shown in the image), and the digestion effect was verified by agarose gel electrophoresis. The linearized vector digested with enzymes was then recovered from the gel. The linearized vector was ligated in vitro with a DNA fragment encoding Lrrc55-shRNA with sticky ends. The ligation product was transformed into competent E. coli cells. After transformation, the target strain was initially screened using LB medium supplemented with ampicillin (100 µg / mL). Single colonies were picked and amplified, and then the bacterial culture was sent to a professional sequencing company for sequencing verification. The sequencing results showed that the fragment inserted in the vector was completely consistent with the target coding sequence of Lrrc55-shRNA (as shown in the image). Figure 2 As shown in the figure, the absence of base deletions, mutations, and mismatches indicates that the recombinant plasmid was successfully constructed. Subsequently, the plasmid of the positive strain was extracted using a nucleic acid extraction kit to obtain the recombinant adeno-associated virus expression vector pENN-AAV-U6-sh-Lrrc55 containing the coding sequence of a short hairpin RNA that targets and knocks down Lrrc55.

[0042] (2) Virus packaging Virus packaging was performed using a three-plasmid packaging system. The constructed target plasmid pENN-AAV-U6-sh-Lrrc55, along with the AAV2 / 9 serotype plasmid and pAdDeltaF6 helper plasmid, were prepared at a dosage of 10 μg / 10 cm culture dish. The required volume of each plasmid was calculated based on the number of 293T cell culture dishes. Simultaneously, the amount of PEI transfection reagent was calculated to be 3-4 times the total plasmid mass. Transfection solution was prepared using empty DMEM medium as a substrate. The three plasmids and PEI transfection reagent were added sequentially, and empty DMEM was added to a final volume of 1 mL / 10 cm culture dish. The dish was vortexed for 30 seconds and then incubated at room temperature for 15 minutes. The transfection solution was then added dropwise along the inner wall of the culture dish to 293T cell culture dishes with 80%–90% confluence. The dishes were incubated at 37°C with 5% CO2. After 10 hours of transfection, the culture medium was replaced with complete medium containing 10% FBS and 1% penicillin-streptomycin for further incubation.

[0043] (3) Virus collection, purification and titer determination Forty-eight hours after medium replacement, cells were observed under a fluorescence microscope. Once >95% of cells showed green fluorescence, cells and culture supernatant were collected into centrifuge tubes. The tubes were centrifuged at 4°C and 4000 rpm for 30 min. The supernatant was then added to PEG8000 at a 1:5 volume ratio, and the mixture was rotated at 4°C for 1 h, followed by standing for 3 h. The mixture was centrifuged again at 4°C and 4000 rpm for 30 min. The supernatant was discarded, and the precipitate was resuspended in lysis buffer. Nuclease and 1 mM MgCl2 were added at a 1:1000 volume ratio, and the mixture was incubated at 37°C for 1 h. The supernatant was then collected by centrifugation to obtain the crude virus extract. Iodixanol was layered in ultracentrifuge tubes at concentrations of 60%, 40%, 25%, and 17%. The crude virus extract was slowly added along the tube wall to the top layer. After ensuring the tubes were properly balanced, the tubes were ultracentrifuged at 4°C and 60,000 rpm. After centrifugation, the virus-rich layer (40% iodixanol) was aspirated using a long needle and concentrated to 1 mL via ultrafiltration. Viral DNA was extracted from 5 μL of the concentrated viral solution and diluted 10-fold to prepare serially diluted standard quality plasmids. Viral titers were determined by quantitative real-time PCR, and the final AAV9-sh-Lrrc55 adeno-associated virus titer was 1.0 × 10⁻⁶. 13 The virus solution was aliquoted at vg / ml and then frozen at -80°C.

[0044] 2. Construction and packaging of AAV9-scramble adeno-associated virus vector Following the complete construction, packaging, purification, and titer determination process of AAV9-sh-Lrrc55, an oligonucleotide fragment encoding a scramble-negative control shRNA sequence without gene targeting was designed and synthesized. Using the same pENN-AAV-U6-shRLuc-eGFP as the base vector, the fragment was digested with restriction endonucleases, ligated in vitro with the oligonucleotide fragment encoding the scramble-shRNA sequence, transformed into *E. coli*, screened in antibiotic-free LB medium, and sequenced for verification. After confirming that the inserted fragment sequence completely matched the expected sequence, plasmids were extracted to obtain the recombinant control vector AAV9-scramble. Subsequently, using the same three-plasmid packaging system, 293T cell transfection conditions, virus collection and purification methods, and quantitative real-time PCR titer determination, the AAV9-scramble virus was packaged and identified. The final AAV9-scramble adeno-associated virus titer was 1.3 × 10⁻⁶. 13 vg / ml, dispensed and frozen at -80℃ for later use.

[0045] Example 2 1. Mouse grouping and model establishment The experimental mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (mouse strain: C57BL / 6J; age: 11 weeks; sex: male) and were randomly divided into two groups: myocardial ischemia-reperfusion injury (IRI) group (experimental group) and control group (Sham group).

[0046] The experimental group conducted a myocardial ischemia-reperfusion injury model construction procedure. Mice were first anesthetized by intraperitoneal injection of 4wt% chloral hydrate prepared with 1×PBS at a dose of 10μl / g. The anesthetized mice were fixed on the operating table, and the hair in their neck and left chest area was removed. Then, under a stereomicroscope, the skin, muscles and connective tissue on the surface of the trachea in the neck of the mice were separated to fully expose the trachea. After that, a small incision was made between the two tracheal cartilage rings below the glottis, and the endotracheal tube was inserted and fixed. The rise and fall of the mouse's chest was observed throughout the process to ensure normal ventilation of both lungs. Next, with the aid of a stereoscope, a transverse incision was made at the fourth and fifth intercostal spaces on the left sternal border of the mouse. The chest wall muscles and intercostal muscles were gently and bluntly separated with microforceps to fully expose the heart. The left anterior descending branch of the heart was ligated. After completion, the thoracic cavity was sutured layer by layer. After 30 minutes of ligation, the mouse's thoracic cavity was reopened, the ligation sutures were cut and removed to restore normal blood supply to the heart. Finally, the intercostal muscles, chest wall muscles and skin were sutured in sequence, and the surgical wound was thoroughly disinfected with iodine.

[0047] The control group underwent sham surgery, with all procedures identical to the experimental group except for the omission of the ligation of the left anterior descending artery. Both groups of mice were closely monitored post-surgery for respiratory distress or airway obstruction. Once fully recovered, the mice were transferred from their warming mats to their regular cages for routine care.

[0048] 2. Injection of the virus via the tail vein The mice in the aforementioned experimental group (IRI group) were further divided into two groups, named AAV9-scramble+IRI group (also labeled AAV9-scramble+AIRI group) and AAV9-sh-Lrrc55+IRI group (also labeled AAV9-sh-Lrrc55+AIRI group); the mice in the control group (Sham group) were also divided into two groups in the same manner, named AAV9-scramble+Sham group and AAV9-sh-Lrrc55+Sham group. Three weeks before the surgical procedure in step 1, all mice in each group were treated with tail vein injection of the virus. Specifically, the mice in each group were placed on a tail vein injection device, and the tails of the mice were thoroughly disinfected with alcohol. Then, using a disposable insulin syringe, 1×10⁻⁶ insulin was injected into each mouse via tail vein injection. 12 The mice were injected with a dose of viral solution, specifically the AAV9-sh-Lrrc55 virus prepared in Example 1 into the AAV9-sh-Lrrc55+IRI and AAV9-sh-Lrrc55+Sham groups, and the AAV9-scramble+IRI and AAV9-scramble+Sham groups into an equal dose of the AAV9-scramble control virus prepared in Example 1. After injection, hemostasis was achieved by gently pressing the injection site on the mouse tail with absorbent cotton, and the mice were then returned to their cages for routine rearing. Three weeks after viral injection, the mice in the AAV9-scramble+IRI and AAV9-sh-Lrrc55+IRI groups underwent myocardial ischemia-reperfusion injury surgery, as described in step 1 above, while the mice in the AAV9-scramble+Sham and AAV9-sh-Lrrc55+Sham groups underwent sham surgery. All mice were anesthetized and euthanized 24 hours (acute myocardial ischemia-reperfusion injury (AIRI)) or 3 weeks (chronic ischemia-reperfusion injury (IRI)) after the corresponding surgery, and heart tissue samples were then taken for the detection and analysis of relevant indicators in the following experimental cases.

[0049] Experimental Example 1 Detection of relative expression levels of Lrrc55 gene and detection of cardiomyocyte apoptosis and necrosis-related indicators in different treatment groups of acute myocardial ischemia-reperfusion injury (AIRI): 1. RT-qPCR detection of relative expression levels of the Lrrc55 gene Example 2: 24 hours after the IRI or sham surgery, mouse heart tissue samples were collected. Total RNA was extracted from the myocardial tissue using the Trizol method, and the total RNA was reverse transcribed into cDNA using a reverse transcription kit. Using the cDNA as a template, real-time quantitative polymerase chain reaction (qPCR) was performed in an iTaq Universal SYBR Green Supermix instrument to detect the relative expression level of the Lrrc55 gene. 18S was used as an internal control gene. -ΔΔCt The relative expression level of the Lrrc55 gene was calculated using a method with 7 replicates for all qPCR reactions.

[0050] Figure 3 To validate the efficiency of AAV9-sh-Lrrc55 knockdown in animals, compared to the AAV9-scramble+Sham group, the relative expression level of Lrrc55 gene in the heart tissue of mice in the AAV9-sh-Lrrc55+Sham group was significantly downregulated, while the expression level of Lrrc55 gene in the AAV9-scramble+AIRI group was significantly increased. The AAV9-sh-Lrrc55+AIRI group significantly reversed the IRI-induced high expression of Lrrc55 gene. All these differences were highly statistically significant. (p<0.001), confirming that AAV9-sh-Lrrc55 can effectively knock down the expression of the Lrrc55 gene in myocardial tissue in vivo.

[0051] 2. Detection of cardiomyocyte apoptosis rate using TUNEL and α-actinin co-staining After the treatment in Example 2, 24 hours after the IRI or sham surgery, mice were anesthetized and euthanized. Heart tissue samples were transversely sectioned and quickly immersed in OCT embedding medium, then frozen at -80°C. Frozen sections with a thickness of 5 μm were prepared using a cryostat. After drying, the sections were co-stained with TUNEL and α-actinin. TUNEL staining was used to label apoptotic cells, α-actinin staining was used to label cardiomyocytes, and DAPI staining was used to label cell nuclei. After staining, the sections were mounted with 50% glycerol to protect from light. Images were observed and acquired using a laser confocal microscope, and the cardiomyocyte apoptosis rate (number of TUNEL-positive cardiomyocytes / total number of cardiomyocytes × 100%) was calculated using ImageJ software.

[0052] The results are as follows Figure 4As shown in the figure. The results showed that compared with the Sham group, the apoptosis rate of cardiomyocytes in the AIRI group was significantly increased; compared with the AAV9-scramble+AIRI group, the apoptosis rate of cardiomyocytes in the AAV9-sh-Lrrc55+AIRI group was significantly decreased, and the differences were highly statistically significant. (p < 0.001), while there was no significant difference between AAV9-scramble and AAV9-sh-Lrrc55 treatments in the Sham group. This indicates that knocking down Lrrc55 can significantly reduce the apoptosis rate of cardiomyocytes after myocardial ischemia-reperfusion injury and improve cardiomyocyte apoptosis injury.

[0053] 3. Lactate dehydrogenase (LDH) activity assay to detect the degree of cardiomyocyte necrosis. Following the treatment in Example 2, 24 hours after the IRI or sham surgery, mice were anesthetized and euthanized. Orbital venous blood samples were immediately collected and incubated overnight at 4°C. After coagulation, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant serum was separated. Using a lactate dehydrogenase (LDH) activity assay kit, following the kit's instructions, the serum samples were appropriately diluted, and LDH activity was measured to quantitatively analyze the degree of cardiomyocyte necrosis.

[0054] The results are as follows Figure 5 As shown, serum LDH activity in mice in both the AAV9-scramble+Sham and AAV9-sh-Lrrc55+Sham groups remained at low levels, with no significant difference between the two groups. Compared with the AAV9-scramble+Sham group, serum LDH activity in mice in the AAV9-scramble+AIRI group was significantly increased, reflecting severe cardiomyocyte necrosis caused by myocardial ischemia-reperfusion injury. Conversely, serum LDH activity in mice in the AAV9-sh-Lrrc55+AIRI group was significantly decreased compared to the AAV9-scramble+AIRI group, with a statistically significant difference. This indicates that knocking down Lrrc55 can effectively reduce the degree of cardiomyocyte necrosis after acute myocardial ischemia-reperfusion injury.

[0055] Experimental Example 2 Lrrc55 gene expression and cardiac function assays after chronic myocardial ischemia-reperfusion injury (IRI): 1. Detection of relative expression levels of Lrrc55 gene in different treatment groups after chronic myocardial ischemia-reperfusion injury Three weeks after the completion of the treatment in Example 2, i.e., after the IRI surgery or sham surgery, mouse heart tissue samples were collected. The relative expression level of Lrrc55 gene in each treatment group was detected according to the RT-qPCR detection method in Example 1 to verify the long-term knockdown effect of AAV9-sh-Lrrc55 on Lrrc55 gene after chronic myocardial ischemia-reperfusion injury.

[0056] The results are as follows Figure 6 As shown, compared to the AAV9-scramble+Sham group, the relative expression level of Lrrc55 gene in the heart tissue of mice in the AAV9-sh-Lrrc55+Sham group was significantly downregulated; the expression level of Lrrc55 gene in mice in the AAV9-scramble+IRI group was higher than that in the Sham group, while the AAV9-sh-Lrrc55+IRI group significantly knocked down the expression of Lrrc55 gene in myocardial tissue after IRI, and the differences were all highly statistically significant. This confirms that AAV9-sh-Lrrc55 can achieve a long-term and stable knockdown effect on the Lrrc55 gene in myocardial tissue in vivo, and can reverse the abnormally high expression of Lrrc55 gene induced by IRI.

[0057] 2. Echocardiography to detect cardiac function in mice Three weeks after the treatment in Example 2, i.e., three weeks after the IRI surgery or sham surgery, the mice were placed in a sealed glass box and anesthetized with 1.5%–2% isoflurane inhalation. Cardiac function was assessed using a Visual Sonics 2100 small animal echocardiography system at a frequency of 30 MHz. Left ventricular long-axis images and corresponding M-mode images were acquired. Left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) were measured and calculated. Each indicator was measured nine times, and the average value was used as the final result to evaluate the mouse's cardiac contractile function.

[0058] The results are as follows Figure 7 As shown, in the Sham group, there was no significant difference in EF between mice treated with AAV9-scramble and AAV9-sh-Lrrc55 (ns indicates no significant difference), but FS was reduced. Compared with the AAV9-scramble+Sham group, the AAV9-scramble+IRI group showed significantly reduced EF and FS, indicating that myocardial ischemia-reperfusion injury led to a severe decline in cardiac function in mice. Conversely, compared with the AAV9-scramble+IRI group, the AAV9-sh-Lrrc55+IRI group showed significantly increased EF and FS, with highly statistically significant differences. This indicates that knocking down Lrrc55 can significantly improve cardiac contractile function in mice after chronic myocardial ischemia-reperfusion injury and inhibit the decline in cardiac function induced by IRI.

[0059] Experimental Example 3 Masson staining to detect the degree of cardiac fibrosis in mice Three weeks after the completion of the treatment in Example 2 (i.e., the IRI surgery or sham surgery), mice were anesthetized and euthanized. Heart tissue samples were extracted, and the heart tissue was transversely sectioned and fixed in 4% paraformaldehyde solution for 48 hours. After fixation, the tissue was dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin to prepare paraffin sections with a thickness of 5 μm. After dewaxing and hydration, the sections were stained using the Masson's trichrome staining kit. Collagen fibers were stained blue, and cardiomyocytes were stained red. After staining, the sections were mounted with neutral resin, observed and images were acquired using a bright-field microscope, and the percentage of fibrosis in the heart tissue was quantitatively analyzed using ImageJ software (area of ​​blue collagen fibers / total myocardial tissue area × 100%).

[0060] The results are as follows Figure 8 As shown, in the Sham group, only a small amount of collagen fibers were observed in the cardiac tissue of mice treated with AAV9-scramble and AAV9-sh-Lrrc55, with an extremely low fibrosis rate, and there was no significant difference between the two groups. Compared with the AAV9-scramble+Sham group, the AAV9-scramble+IRI group showed a significantly higher fibrosis rate in the cardiac tissue, indicating that myocardial ischemia-reperfusion injury induced severe cardiac fibrosis. Conversely, compared with the AAV9-scramble+IRI group, the AAV9-sh-Lrrc55+IRI group showed a significantly lower fibrosis rate in the cardiac tissue, with a highly statistically significant difference. This indicates that knocking down Lrrc55 can effectively alleviate cardiac fibrosis induced by myocardial ischemia-reperfusion injury and inhibit the progression of pathological ventricular remodeling.

[0061] In summary, this invention demonstrates that knocking down the expression of the Lrrc55 gene can significantly reduce cardiomyocyte apoptosis and necrosis after myocardial ischemia-reperfusion, alleviate cardiac fibrosis, improve pathological ventricular remodeling, and effectively enhance cardiac contractile function. This discovery provides a novel direction for drug development in the prevention and treatment of myocardial ischemia-reperfusion injury and heart failure, clarifies the important value of Lrrc55 as a potential drug target, and has significant theoretical implications and clinical application prospects.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Lrrc55 inhibitors in the preparation of drugs for the prevention and treatment of myocardial ischemia-reperfusion injury.

2. The application of Lrrc55 inhibitors in the preparation of drugs for the prevention and treatment of any of the following diseases, characterized in that: (1) Cardiac cell apoptosis; (2) Cardiac cell necrosis; (3) Cardiac fibrosis; (4) Decreased cardiac function; (5) Pathological ventricular remodeling after myocardial ischemia-reperfusion; (6) Heart failure.

3. The application according to claim 1 or 2, characterized in that, The Lrrc55 inhibitor includes nucleic acid molecules or reagents capable of downregulating the expression level of the Lrrc55 gene; or includes reagents capable of inhibiting the activity of the Lrrc55 protein.

4. The application according to claim 3, characterized in that, The nucleic acid molecule includes at least one of small interfering RNA, short hairpin RNA, microRNA, and antisense oligonucleotide that targets the Lrrc55 gene.

5. The application according to claim 4, characterized in that, The short hairpin RNA is transcribed in a host cell from a coding sequence comprising the positive-strand nucleotide sequence shown in SEQ ID NO.1 and the anti-strand nucleotide sequence shown in SEQ ID NO.

2.

6. The application according to claim 3, characterized in that, The reagent capable of downregulating Lrrc55 gene expression includes a recombinant adeno-associated virus vector carrying an Lrrc55-shRNA coding sequence; the Lrrc55-shRNA coding sequence includes the positive-strand nucleotide sequence shown in SEQ ID NO.1 and the anti-strand nucleotide sequence shown in SEQ ID NO.2; the serotype of the recombinant adeno-associated virus vector is AAV9.

7. A drug for preventing and / or treating myocardial ischemia-reperfusion injury, characterized in that, The drug can inhibit the expression of the Lrrc55 gene or Lrrc55 protein.

8. The medicament according to claim 7, characterized in that, The active ingredient of the drug includes short hairpin RNA, which is transcribed in host cells from a coding sequence, the coding sequence including the positive-strand nucleotide sequence shown in SEQ ID NO.1 and the anti-strand nucleotide sequence shown in SEQ ID NO.

2.

9. A method for screening drugs to prevent and treat myocardial ischemia-reperfusion injury, characterized in that, The effect of the test drug on the expression level of the Lrrc55 gene or its protein was detected. If the test drug can reduce the expression level of the Lrrc55 gene or its protein, it indicates that the test drug has a potential role in preventing and treating myocardial ischemia-reperfusion injury.

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