Application of microRNA 244-5P inhibitor in preparation of product for treating sepsis cardiomyopathy

Abstract

The invention relates to the technical field of medicine preparation, in particular to application of a microRNA244-5P inhibitor in preparation of a product for treating sepsis cardiomyopathy. According to the application of the microRNA 244-5P inhibitor in the preparation of the product for treating the sepsis cardiomyopathy, the sequence of the microRNA 244-5P inhibitor is shown as Seq. ID No. 1, and the microRNA 244-5P inhibitor can be applied to the preparation of the product for treating the sepsis cardiomyopathy. The provided microRNA 244-5P inhibitor lays a precise foundation for targeted treatment of sepsis cardiomyopathy. The inhibitor can specifically recognize and act on related targets, accurately intervene the pathological process of sepsis cardiomyopathy, has more stable structure and activity, can ensure that the inhibition effect can be continuously and efficiently played when a treatment product is prepared, provides a clear material basis and a technical basis for research, development, production and quality control of subsequent products, and has a wide application prospect. The pertinence and the effectiveness of treatment are greatly improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine preparation, and particularly relates to application of a microRNA 244-5P inhibitor in preparation of a product for treating sepsis cardiomyopathy. BACKGROUND

[0002] Sepsis, a critical illness caused by the dysregulation of host response to infection, is one of the leading causes of death in intensive care units (ICU) and affects millions of patients worldwide each year. Its severe complication, sepsis cardiomyopathy (also known as sepsis cardiac dysfunction), directly determines the prognosis of patients and becomes an important driver of sepsis-related deaths. Pathologically, the excessive inflammatory response triggered by sepsis leads to the release of a large number of myocardial suppressors, forming an inflammatory factor storm. The abnormal upregulation of pro-inflammatory cytokines represented by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) directly damages myocardial cells, interferes with energy metabolism and destroys myocardial contractile function, ultimately leading to cardiac dysfunction.

[0003] MicroRNAs (miRNAs) are a class of non-coding single-stranded RNAs with a length of about 22 nucleotides, which regulate gene expression at the post-transcriptional level by recognizing the 3' untranslated region of target mRNA, mediating translation inhibition or mRNA degradation. Their high conservation and stability make them core regulators of various physiological and pathological processes. Recent studies have revealed that miRNAs play multiple regulatory roles in sepsis cardiomyopathy: on the one hand, they regulate programmed cell death of myocardial cells by targeting apoptosis-related genes such as the Bcl-2 family and caspase pathway; on the other hand, they participate in the cascade reaction of inflammatory signaling pathways such as NF-kappa B and MAPK, affecting the balance of pro-inflammatory / anti-inflammatory; in addition, exosome-mediated miRNA transmission can exert protective effects across cells, such as improving myocardial cell damage by transporting anti-apoptotic or antioxidant-related miRNAs. These mechanisms suggest that the miRNA network is closely related to the occurrence and development of sepsis myocardial injury, but the spatiotemporal expression characteristics and key target genes in the disease process still need to be further explored.

[0004] Current treatment of sepsis cardiomyopathy is limited to symptomatic support, while the discovery of miRNAs provides a new perspective for precision medicine. Recent studies have shown that miRNAs play an important role in treating sepsis cardiomyopathy by regulating apoptosis-related genes of myocardial cells, affecting inflammatory responses and cell signaling pathways, and protecting myocardial cells through exosome-mediated miRNA transmission. However, the highly relevant miRNAs for sepsis cardiomyopathy have not been fully explored. SUMMARY

[0005] The application aims to provide an application of a microRNA 244-5P inhibitor in the preparation of a product for treating sepsis cardiomyopathy, and aims to solve the problem that there is no miRNA highly related to sepsis cardiomyopathy in the prior art.

[0006] To achieve the above application purposes, the technical scheme adopted by the application is as follows:

[0007] In the first aspect, the application provides an application of a microRNA 244-5P inhibitor in the preparation of a product for treating sepsis cardiomyopathy, and the sequence of the microRNA 244-5P inhibitor is shown in Seq.ID No. 1.

[0008] In some embodiments, the product loads the microRNA 244-5P inhibitor through tea leaf exosomes.

[0009] In some embodiments, the tea leaf exosomes are green tea exosomes, and the particle size of the green tea exosomes is 124nm-125.6nm.

[0010] In some embodiments, the microRNA 244-5P inhibitor improves sepsis cardiomyopathy by inhibiting ferroptosis of myocardial cells, and at least one of the following performances is shown:

[0011] Lowering the level of intracellular reactive oxygen species;

[0012] Up-regulating the expression of GPX4;

[0013] Down-regulating the expression of PTGS2.

[0014] In some embodiments, the product includes a drug.

[0015] In the second aspect, the application provides a pharmaceutical composition for treating sepsis cardiomyopathy, and the pharmaceutical composition includes a microRNA 244-5P inhibitor, and the sequence of the microRNA 244-5P inhibitor is shown in Seq.ID No. 1.

[0016] In some embodiments, the pharmaceutical composition further includes tea leaf exosomes, wherein the tea leaf exosomes are used to load the microRNA 244-5P inhibitor.

[0017] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0018] In some embodiments, the pharmaceutically acceptable excipient includes a stabilizer, a protective agent, a preservative, and a bacteriostatic agent.

[0019] In some embodiments, the dosage form of the pharmaceutical composition includes any one of an injection, an oral agent, a gel, a spray, a patch, a plaster, and a tablet.

[0020] The microRNA 244-5P inhibitor provided in the first aspect of the present application lays a precise foundation for targeted treatment of sepsis cardiomyopathy. The inhibitor can specifically recognize and act on the relevant target, and precisely intervene in the pathological process of sepsis cardiomyopathy. Compared with the inhibitor with an unclear sequence, the inhibitor with a specific sequence has a more stable structure and activity, which can ensure that the inhibitory effect is continuously and efficiently exerted when preparing a treatment product, and provides a clear material basis and technical basis for subsequent product research, production and quality control, greatly improving the targeting and effectiveness of treatment.

[0021] The pharmaceutical composition provided in the second aspect of the present application contains the microRNA 244-5P inhibitor with a specific sequence, and forms a synergistic treatment system by reasonably compounding the inhibitor with other ingredients. Compared with a single component, the pharmaceutical composition can exert the synergistic effect of multiple components, not only enhancing the treatment effect on sepsis cardiomyopathy, but also reducing the dosage of a single component and reducing potential adverse reactions. At the same time, the pharmaceutical composition is more convenient for dosage form development and optimization, and can adjust the proportion and dosage form of each component according to different treatment needs and administration routes, improve the stability and bioavailability of the drug, and provide more flexible and effective treatment options for clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0023] Figure 1 is an analysis graph of mouse heart ultrasound provided by the embodiments of the present application.

[0024] Figure 2 is an EF / FS statistical analysis graph provided by the embodiments of the present application.

[0025] Figure 3 is a heart cell HE staining graph provided by the embodiments of the present application.

[0026] Figure 4 is an ELISA analysis graph of plasma GPX4, PTGS2 and troponin after 24 hours of LPS modeling provided by the embodiments of the present application.

[0027] Figure 5 is a survival rate analysis graph of mice provided by the embodiments of the present application.

[0028] Figure 6is a ROS level analysis chart in a cell experiment provided by an embodiment of the present application.

[0029] Figure 7 is a ROS level analysis chart in a cell experiment provided by an embodiment of the present application.

[0030] Figure 8 is a GPX4 and PTGS2 level analysis chart in a cell experiment provided by an embodiment of the present application.

[0031] Figure 9 is a microelectroscope analysis chart in a cell experiment provided by an embodiment of the present application. DETAILED DESCRIPTION

[0032] In order to make the technical problems to be solved by the present application, the technical solutions and beneficial effects clearer, the present application will be further described in detail below with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0033] In the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0034] In the present application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions means any combination of these items, including any combination of single item or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can mean a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple.

[0035] It should be understood that in various embodiments of the present application, the size of the sequence number of the above processes does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0036] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0037] The weight of the related components mentioned in the embodiment specification of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of each component. Therefore, as long as the content of the related components in the embodiment specification of the present application is proportionally enlarged or reduced, it is within the scope disclosed in the embodiment specification of the present application. Specifically, the mass in the embodiment specification of the present application can be μg, mg, g, kg, etc. mass units commonly known in the chemical industry.

[0038] The terms "first", "second" are only for descriptive purposes, to distinguish the objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. For example, without departing from the scope of the present application, the first XX can also be referred to as the second XX, and similarly, the second XX can also be referred to as the first XX. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features.

[0039] The first aspect of the embodiment of the present application provides the use of a microRNA 244-5P inhibitor in the preparation of a product for treating sepsis cardiomyopathy. The sequence of the microRNA 244-5P inhibitor is shown in Seq. ID No. 1.

[0040] The microRNA 244-5P inhibitor provided by the first aspect of the embodiment of the present application lays a precise foundation for targeted treatment of sepsis cardiomyopathy. The inhibitor can specifically recognize and act on the related target, and precisely intervene in the pathological process of sepsis cardiomyopathy. Compared with the inhibitor with an unclear sequence, the inhibitor with this specific sequence has a more stable structure and activity, which can ensure that it can continuously and efficiently exert an inhibitory effect when preparing a treatment product, and provides a clear material basis and technical basis for subsequent product research, production and quality control, greatly improving the targeting and effectiveness of treatment.

[0041] In some specific embodiments, the sequence of the microRNA 244-5P inhibitor is shown in Seq. ID No. 1, and Seq. ID No. 1 is specifically CUAAACGGAACCACUAGUGACUUGA. Among them, the full chain of the microRNA 244-5P inhibitor is modified by methoxy, there are 2 and 4 thio skeleton modifications at the 5' end and the 3' end respectively, and a high affinity cholesterol modification is connected at the 3' end.

[0042] The sequence is synthesized by Biosynthetic Corporation, and the specific information of the synthesized sequence is shown in the following table:

[0043]

[0044] In some embodiments, the product is loaded with microRNA 244-5P inhibitor by tea leaf exosomes. The use of tea leaf exosomes loaded with microRNA 244-5P inhibitor breaks through the limitations of traditional drug delivery. As a natural nanoscale carrier, tea leaf exosomes have excellent biomembrane fusion ability and can carry the inhibitor to smoothly cross the physiological barrier and accurately reach the diseased myocardial cells. This not only improves the enrichment of the inhibitor at the lesion site and enhances the local drug concentration, thereby improving the treatment effect, but also reduces the distribution of the inhibitor in non-target tissues and reduces the potential toxic side effects on normal tissues, effectively improving the safety and targeting of treatment, and providing a more efficient and safe drug delivery scheme for the treatment of sepsis cardiomyopathy.

[0045] In some embodiments, the tea leaf exosomes are green tea exosomes, and the particle size of the green tea exosomes is 124-125.6 nm. Limiting the tea leaf exosomes to green tea exosomes with a particle size of 124-125.6 nm further optimizes the performance of the carrier. Green tea exosomes with this particle size range have unique physical and chemical properties, are not easily removed by macrophages during in vivo circulation, can more stably transport the inhibitor, and prolong its half-life in blood circulation. At the same time, this particle size makes it easier to penetrate the interstitial space and accurately enter the myocardial cells, significantly improving the efficiency of drug delivery. In addition, green tea exosomes are widely available and have a relatively simple preparation process, which is conducive to large-scale production and reduces production costs, providing economic feasibility and technical advantages for the clinical promotion and application of the product.

[0046] In some embodiments, the microRNA 244-5P inhibitor improves sepsis cardiomyopathy by inhibiting myocardial cell ferroptosis, specifically at least one of the following:

[0047] Lowering the level of intracellular reactive oxygen species;

[0048] Up-regulating GPX4 expression;

[0049] Down-regulating PTGS2 expression.

[0050] The specific mechanism of the microRNA 244-5P inhibitor for improving sepsis cardiomyopathy by inhibiting myocardial cell ferroptosis is provided, mainly reducing the intracellular reactive oxygen level, up-regulating GPX4 expression, and down-regulating PTGS2 expression, and the treatment principle is clarified from the molecular level. Reducing the intracellular reactive oxygen level can directly reduce the damage of oxidative stress to myocardial cells and protect the biological macromolecules in cells; up-regulating GPX4 expression can enhance the antioxidant capacity of cells, maintain the integrity of the structure and function of the cell membrane, and effectively prevent the occurrence of ferroptosis; down-regulating PTGS2 expression can block the abnormal inflammatory response and the activation of the ferroptosis signal pathway, and the multi-target synergistic effect can inhibit the ferroptosis process of myocardial cells from the root, providing a scientific and effective action target and theoretical basis for the treatment of sepsis cardiomyopathy, and significantly improving the scientificity and effectiveness of the treatment.

[0051] In some embodiments, the product includes a drug. The explicit inclusion of the drug in the product broadens the application range of the microRNA 244-5P inhibitor, enabling it to be directly applied to the field of clinical treatment. As a treatment product directly acting on patients, the drug can ensure the safety and effectiveness of the product through a strict drug research and development, production, and quality control system. This limitation makes it possible for the microRNA 244-5P inhibitor to be transformed from a laboratory research achievement to a clinical application, providing a new drug treatment option for sepsis cardiomyopathy patients and having important clinical application value and social significance.

[0052] The second aspect of the embodiments of the present application provides a pharmaceutical composition for treating sepsis cardiomyopathy, which comprises a microRNA 244-5P inhibitor, and the sequence of the microRNA 244-5P inhibitor is shown in Seq. ID No. 1.

[0053] The pharmaceutical composition provided by the second aspect of the embodiments of the present application comprises a microRNA 244-5P inhibitor with a specific sequence, and by reasonably compounding the inhibitor with other ingredients, a synergistic treatment system is formed. Compared with a single component, the pharmaceutical composition can exert the synergistic effect of multiple components, not only enhancing the treatment effect on sepsis cardiomyopathy, but also reducing the dosage of a single component and reducing potential adverse reactions. At the same time, the pharmaceutical composition is more convenient for dosage form development and optimization, and the proportion and dosage form of each component can be adjusted according to different treatment needs and administration routes, improving the stability and bioavailability of the drug and providing a more flexible and effective treatment scheme for clinical treatment.

[0054] In some embodiments, the pharmaceutical composition further comprises tea leaf exosomes, wherein the tea leaf exosomes are used to load the microRNA 244-5P inhibitor. The addition of tea leaf exosomes to load the inhibitor in the pharmaceutical composition combines the advantages of the carrier with the synergistic effect of the pharmaceutical composition. Tea leaf exosomes can effectively protect the stability of the inhibitor during in vivo transportation, avoiding its enzymatic hydrolysis or degradation; at the same time, the targeting of exosomes ensures the precise arrival of the inhibitor to the diseased myocardial cells, improving the concentration of the drug at the lesion site. The synergistic effect of other components in the pharmaceutical composition and the exosome-inhibitor can further enhance the therapeutic effect, reduce the treatment cost, and improve the overall performance of the drug, providing a more innovative and practical solution for the treatment of sepsis cardiomyopathy.

[0055] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The pharmaceutical composition contains a pharmaceutically acceptable excipient, which plays a key role in improving the stability, safety and effectiveness of the drug. The stabilizer can prevent the degradation or deterioration of the inhibitor during storage and use, ensuring the stability of the content of the active ingredients of the drug; the protective agent can maintain the physicochemical properties of the drug, avoiding its influence by external environmental factors; the preservative and bacteriostatic agent can effectively inhibit the growth and reproduction of microorganisms, prevent the contamination of the drug, and ensure the safety and quality controllability of the drug. The addition of these excipients makes the pharmaceutical composition more in line with the requirements of drug production and clinical application, prolongs the shelf life of the drug, and improves the safety and effectiveness of the drug.

[0056] In some embodiments, the pharmaceutically acceptable excipient comprises a stabilizer, a protective agent, a preservative, and a bacteriostatic agent. The specific limitation of the type of pharmaceutically acceptable excipient provides clear guidance for the formulation design and production of the pharmaceutical composition. Different types of excipients have their own unique functions and mechanisms of action, and reasonable selection and collocation of these excipients can maximize the optimization of the performance of the pharmaceutical composition. For example, a suitable stabilizer can significantly improve the chemical stability of the inhibitor, a protective agent can enhance the environmental tolerance of the drug, and a preservative and bacteriostatic agent can ensure the microbial safety of the drug during storage and use. This precise limitation of the type of excipient helps to improve the standardization and standardization of drug production, ensures the consistency and stability of drug quality, and provides reliable protection for the industrialized production and clinical application of the product.

[0057] In some embodiments, the dosage form of the pharmaceutical composition includes any one of injections, oral agents, gels, sprays, patches, ointments, tablets. The rich selection of dosage forms fully considers the needs of different patient groups and clinical treatment scenarios. Injections have the characteristics of rapid onset and high bioavailability, and are suitable for patients with critical conditions who need to quickly control symptoms; oral agents are convenient to take and suitable for patients who need long-term home treatment, which can improve patient medication compliance; topical dosage forms such as gels, sprays, patches, and ointments can achieve local drug delivery, reduce systemic adverse reactions, and are suitable for treatment of specific parts. The diversified dosage forms provide clinicians with more treatment options and choices, enabling the development of personalized treatment plans based on the specific condition, age, physical condition and medication preferences of patients, significantly improving the flexibility and adaptability of treatment, and meeting the treatment needs of different patients.

[0058] The following will be described in conjunction with specific embodiments.

[0059] Example 1

[0060] Preparation of exosome loaded with miR-244-5P inhibitor

[0061] Using Exoload small nucleic acid loading kit (Cat#ELSR-06-02, echo biotech, China), first add miR-244-5P inhibitor 1000 pmol, then add 3×10 11 The tea exosome particles were added with 200 μL of exosome transport peptide and 5 μL of reaction solution to form a loading mixture, and the mixture was incubated at 37°C on a shaker (constant speed 150 rpm / min) for 2 h in the dark.

[0062] Take 50 μL of loaded tea exosomes, dilute to 1.5 ml with PBS, filter with a 0.22 μm needle filter (BS-PES25-22-S, biosharp), and then detect with a multi-laser nanoparticle tracking analysis system (NTA) to obtain exosomes loaded with miR-244-5P inhibitor.

[0063] Performance testing and result analysis

[0064] (1) Provide a mouse model with sepsis

[0065] Preparation method of sepsis mouse model: 8-12 week-old SPF male C57BL / 6 wild type purchased from Guangzhou Jinwei Biotechnology Co., Ltd. Animals were studied at 8-12 weeks of age and 25-30 g of body weight. Sepsis was induced by intraperitoneal injection of 12 mg / kg LPS. The control group was injected with the same dose of normal saline to rule out errors. 12 h later, the loaded exosomes were injected into the tail vein of the mouse. 24 h later, the mouse infection was observed, and the cardiac function was determined by echocardiography. Another batch of mice was also euthanized by cervical dislocation 24 h later. The plasma and heart were collected, and the myocardial injury was observed by HE staining of the heart section. The plasma GPX4, PTGS2, and troponin were determined by ELISA for analysis.

[0066] (ii) In vivo animal experiment analysis

[0067] 1. Heart B-ultrasound analysis

[0068] Experimental steps of cardiac ultrasound: First, the mouse was anesthetized with a gas anesthetic. After anesthesia, the mouse limbs were coated with conductive glue, and the mouse was fixed on the monitoring table with adhesive tape. Then the mouse chest hair was removed with depilatory cream, and then washed with water. When the mouse heart rate was stable at 400-500 times / min, the probe was used to obtain the cardiac ultrasound data. Four to five cardiac cycles of M-type small animal echocardiogram records were obtained under two-dimensional guidance. The software semi-automatically measured the HR (heart rate), LVESD (left ventricular end-systolic diameter), LVEDD (left ventricular end-diastolic diameter), LVESV (left ventricular end-systolic volume), LVEDV (left ventricular end-diastolic volume), SV (stroke volume), CO (cardiac output), LV Mass (left ventricular myocardial weight), LVAW;s (systolic left ventricular anterior wall thickness), LVAW;d (diastolic left ventricular anterior wall thickness), LVPW;s (systolic left ventricular posterior wall thickness), LVPW;d (diastolic left ventricular posterior wall thickness), and other data.

[0069] To simulate the clinical sepsis treatment process, the loaded exosomes were injected into the mouse body through the tail vein 12 h after modeling. Similarly, the same volume of PBS was injected into the mouse body. 24 h later, the heart was ultrasonicated. According to the results of echocardiography, as shown in Figure 1 , it can be found that, as shown in Figure 2 , the cardiac EF value decreased from 77.36% to 45.5%. After inhibition of miR-244-5P, the cardiac systolic and diastolic functions were significantly restored. The EF value of the LPS+miR-244-5P inhibitor group increased to 69.43% (as shown in echocardiogram and EF / FS statistical graph).

[0070] 2. Heart cell HE staining

[0071] Heart cell HE staining experimental procedure: 12 hours after intraperitoneal injection of LPS in mice, tail vein injection of loaded exosomes, 24 hours later, the mouse spinal cord was dislocated and killed, the chest was opened with scissors, and the heart apex was carefully punctured with a 1 ml syringe needle. 20-30 ml of PBS was used to perfuse the heart apex, and the whole heart was white. The heart can be removed, the fat tissue around the heart is removed with forceps, and the heart is soaked in 4% paraformaldehyde. Mark the group and date, and send to Wuhan Baiji Biological Technology Co., Ltd. After HE section preparation is completed, the microscope is used for observation, and the image is collected and analyzed.

[0072] As shown in Figure 3 , heart HE staining also showed the same trend. The myocardial fibers of the LPS group were arranged irregularly, the interstitial edema was reduced, and the inflammatory cell infiltration was inhibited. After inhibition of miR-244-5P, the myocardial fibers returned to normal arrangement, and the interstitial edema was reduced.

[0073] 3. ELISA experiment (providing plasma after modeling for 24h, ELISA experiment analysis of GPX4, PTGS2 and troponin)

[0074] ELISA: GPX4 (MM-44846M1, enzyme immune, China), PTGS2 (MM-45369M1 enzyme immune, China), TNNI3 / cTn-I (E-EL-M1203, Elabscience, China), after modeling for 24h, the mouse peripheral blood was collected by enucleation method in EDTA anticoagulant tube, centrifuged at 4°C, 3000rpm for 15min, and the upper plasma was collected. Immediately detect or store at-80℃, then use ELISA kit analysis according to the instruction steps, detect the level of GPX4, PTGS2, TNNI3 / cTn-I in mouse plasma.

[0075] As shown in Figure 4 , the plasma after LPS modeling for 24h was collected to do ELISA of GPX4, PTGS2 and troponin, and it was found that the GPX4 of LPS group was down-regulated from 415.6pg / ml to 347.5pg / ml, PTGS2 was up-regulated from 278.8ng / ml to 344.9ng / ml, and the level of troponin was increased from 1.541pg / ml to 659.4pg / ml. After inhibition of miR-244-5P, the GPX4 of LPS+miR-244-5P inhibitor group was up-regulated to 400pg / ml, PTGS2 was down-regulated to 249.1ng / ml, and the level of troponin was reduced to 453pg / ml. Similar to the trend of ferroptosis inhibitor.

[0076] 4. Survival rate

[0077] Survival rate: Mice were injected intraperitoneally with 12 mg / kg of LPS as a sepsis model, and 1 h later, tail vein injection of loaded exosomes or equivalent PBS was performed. The mice were placed in the same suitable environment, and the number of surviving mice was observed every 12 h.

[0078] As shown in Figure 5 , it can be seen that all mice in the LPS group died on the second day, and the survival rate of the miR-244-5P inhibitor group increased to 40%, with a significant effect.

[0079] In summary, the miR-244-5P inhibitor can significantly improve the heart function of sepsis mice and significantly increase the survival rate.

[0080] (III) Analysis of in vitro cell experiments

[0081] 1. Cell culture and treatment

[0082] HL-1 cells were seeded in a six-well culture plate at an appropriate concentration of 100 cells per well and cultured in a culture incubator containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 5% penicillin / streptomycin in MEM medium (Gibco; Thermo Fisher Scientific, Inc.), with a culture condition of 37°C, 5% carbon dioxide. After most of the cells were fixed in the well, the cells were treated with a miR-2445p inhibitor at a concentration of 50 nM for 24 hours, and then stimulated with LPS (20 μg / ml, L8880, Sarlbio) for 12 hours in a culture incubator at 37°C, 5% carbon dioxide.

[0083] 2. Detection of intracellular reactive oxygen species generation

[0084] The HL-1 cells were treated and placed in minimal essential medium containing 10 micromoles of DCFH-DA (a product of Becton Dickinson) and incubated at 37°C for 20 minutes. After washing with PBS for 3 times, the fluorescence intensity of the cells was measured using a flow cytometer.

[0085] 3. Enzyme-linked immunosorbent assay

[0086] Using an ELISA commercial kit, mouse GPX4 (MM-44846M1, Meso Scale Diagnostics) and mouse GPX4 (MM-44846M1, Meso Scale Diagnostics) in serum or HL-1 cell culture supernatant were analyzed according to the manufacturer's protocol.

[0087] 4. Transmission electron microscopy

[0088] HL-1 cells were fixed with 2.5% glycerol solution for 30 minutes, and then placed in a refrigerator at 4°C overnight at room temperature. Next, the cells were dehydrated with alcohol and acetone, and then embedded with epoxy resin for 2 hours at room temperature, and then sectioned with an ultramicrotome (thickness 70-80 nm). These sections were stained with uranium-lead double staining agent (2% uranyl acetate and 2.6% lead citrate) for transmission electron microscopy observation.

[0089] As shown in Figure 6 , pretreatment of miR-2445p inhibitor can effectively reduce the level of reactive oxygen species (ROS) in cells. In the cell experiment, the level of ROS (ROS is the generation of active oxygen in cells, and the addition of LPS group shows that the increase of ROS generation indicates the aggravation of cell damage, and the addition of Micro-RNA2445p group shows improvement.

[0090] As shown in Figure 7 , the study evaluated the generation of intracellular reactive oxygen species (ROS) induced by lipopolysaccharide (LPS) by a method based on the generation of fluorescent DCF by DCFH-DA oxidation dependent on peroxide. The specific operation is to detect the DCF fluorescence intensity after treating the cells with LPS for 12 hours. In the experimental setting, the group without the addition of LPS is used as the negative control, and the group with the addition of LPS and Fer-1 is used as the positive control. The results of the study show that, compared with the control group, the HL-1 cells treated with LPS have a significantly increased ROS production. However, if the cells are pretreated with miR-2445p inhibitor for 24 hours before LPS treatment, the increase in ROS production can be effectively inhibited, and the inhibitory effect is similar to that of the LPS combined with Fer-1 treatment group. After pretreatment with micro-2445p inhibitor, the inhibitory effect of ferroptosis is consistent with that of existing ferroptosis inhibitors on the market. However, under the same experimental conditions, pretreatment with miR-2445p inhibitor negative control (NC) for 24 hours did not observe similar effects.

[0091] To further explore the phenomenon of ferroptosis in HL-1 cells, the cell culture supernatant was separated and detected. As shown in Figure 8As shown, enzyme-linked immunosorbent assay (ELISA) results indicated that glutathione peroxidase 4 (GPX4) levels were significantly decreased in the LPS-treated group, while GPX4 levels were increased in the LPS combined with Fer-1 treatment group. Simultaneously, the prostaglandin internal peroxidase 2 (PTGS2) levels showed opposite trends in the two groups. Notably, 24-hour pretreatment with miR-2445p inhibitors effectively reversed the decrease in GPX4 levels and increase in PTGS2 levels caused by LPS treatment. However, 24-hour pretreatment of the miR-2445p inhibitor negative control (NC) did not significantly affect the experimental results. These results strongly suggest that miR-2445p inhibitors may reduce the pathogenic effects of lipopolysaccharide (LPS) by inhibiting ferroptosis.

[0092] like Figure 9 As shown, transmission electron microscopy (TEM) further revealed that LPS-treated HL-1 cells exhibited a series of typical ferroptosis-related morphological features in their mitochondria, specifically a reduction in the number of mitochondrial cristae, rupture of the outer mitochondrial membrane, and decreased mitochondrial membrane density. In cells pretreated with the miR-2445p inhibitor for 24 hours, these mitochondrial morphological damages were significantly improved.

[0093] In summary, the microRNA 244-5P inhibitor provided in this application lays a precise foundation for targeted therapy of septic cardiomyopathy; specifically, it inhibits ferroptosis in septic cardiomyopathy and improves cardiac function. This inhibitor can specifically recognize and act on relevant targets, precisely intervening in the pathological process of septic cardiomyopathy. Compared to inhibitors with undefined sequences, this specific sequence inhibitor has a more stable structure and activity, ensuring sustained and efficient inhibitory effects during the preparation of therapeutic products. This provides a clear material basis and technical support for subsequent product development, production, and quality control, greatly improving the targetedness and effectiveness of treatment.

[0094] The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Use of a microRNA 244-5P inhibitor for the manufacture of a product for the treatment of sepsis cardiomyopathy, characterized in that, The sequence of the microRNA 244-5P inhibitor is shown as Seq.ID No.

1.

2. Use according to claim 1, characterized in that, The product loads the microRNA 244-5P inhibitor through tea leaf exosomes.

3. Use according to claim 2, characterized in that, The tea leaf exosomes are green tea exosomes, and the particle size of the green tea exosomes is 124nm-125.6nm.

4. Use according to any one of claims 1 to 3, characterized in that, The microRNA 244-5P inhibitor improves sepsis cardiomyopathy by inhibiting myocardial cell ferroptosis, and at least one of the following performances is shown: Lowering the level of intracellular reactive oxygen species; Up-regulating GPX4 expression; Down-regulating PTGS2 expression.

5. Use according to any one of claims 1 to 3, characterized in that, The product comprises a medicine.

6. A pharmaceutical composition for treating sepsis cardiomyopathy, characterized by, The pharmaceutical composition comprises a microRNA 244-5P inhibitor, and the sequence of the microRNA 244-5P inhibitor is shown as Seq.ID No.

1.

7. The pharmaceutical composition for treating sepsis cardiomyopathy according to claim 6, characterized in that, The pharmaceutical composition further comprises tea leaf exosomes, wherein the tea leaf exosomes are used to load the microRNA 244-5P inhibitor.

8. The pharmaceutical composition for treating sepsis cardiomyopathy according to claim 6, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

9. The pharmaceutical composition for treating sepsis cardiomyopathy according to claim 6, wherein The pharmaceutically acceptable adjuvant comprises a stabilizer, a protective agent, a preservative, and a bacteriostatic agent.

10. The pharmaceutical composition for treating sepsis cardiomyopathy according to claim 6, characterized in that, The dosage form of the pharmaceutical composition comprises any one of an injection, an oral agent, a gel, a spray, a patch, a paste, and a tablet.

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