Application of miRNA-149-5p and agonist of miRNA-149-5p in preparation of medicine for treating sepsis lung injury

Abstract

The invention relates to the technical field of medicines, and provides application of miRNA-149-5p in preparation of a medicine for treating sepsis lung injury. An in-vitro cell experiment shows that the miRNA-149-5p agonist is used for treating the vascular endothelial cells, so that the functions of the vascular endothelial cells under the sepsis condition can be saved, including improving the barrier function of the vascular endothelial cells and reducing the permeability of the vascular endothelial cells; the proliferation function is improved; the inflammation level is reduced; the mononuclear cell adhesion is reduced; and the like. In-vivo animal experiments show that miRNA-149-5p agonist treatment can improve the pathological structure of sepsis lung injury, improve pulmonary edema and tissue protein exudation, and improve the pulmonary vessel barrier ability. Therefore, a new basis is provided for the miRNA-149-5p and the agonist of the miRNA-149-5p to promote the repair of the sepsis lung injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to the application of miRNA-149-5p as a target in the treatment of sepsis-induced lung injury, specifically to the application of miRNA-149-5p in the preparation of drugs for treating sepsis-induced lung injury. Background Art

[0002] Sepsis is a clinical syndrome characterized by microvascular dysfunction and immune-inflammatory homeostasis imbalance caused by infection or injury. Among them, the lung is the most susceptible target organ of sepsis, and about 25-50% of sepsis patients may develop acute lung injury or even acute respiratory distress syndrome. Its pathological features include over-activated inflammatory response, alveolar-capillary barrier disruption, pulmonary edema, and refractory hypoxemia. Among them, endothelial barrier function impairment is considered to be the main mechanism of sepsis-induced lung injury. In the highly inflammatory environment of sepsis, endothelial cells are damaged, thus transforming into pro-apoptotic, pro-inflammatory, pro-adhesive, and pro-coagulant phenotypes, resulting in increased vascular permeability, increased transendothelial migration of inflammatory cells, and exudation of red blood cells and proteins, thereby causing tissue edema, etc., seriously affecting lung function.

[0003] MicroRNA (miRNA) is a class of non-coding single-stranded small molecule RNAs with a length of 18-22 nucleotides, which can adsorb to the 3'-UTR region of downstream target genes, resulting in the degradation or translational inhibition of target gene mRNA. Recent studies have shown that various miRNAs play important roles in the occurrence and development of sepsis-induced lung injury. Based on this, exogenous regulation of miRNA expression levels may be a potential strategy for treating sepsis-induced lung injury. Compared with traditional drug therapy, the development of specific miRNA analogs or agonists for treating sepsis-induced lung injury has the following advantages: ① It is relatively easy to prepare and has a faster development process, which is more conducive to clinical translation; ② It can achieve multi-target regulation and coordinately regulate the occurrence and development of diseases; ③ Endogenous miRNAs are usually produced rapidly and degraded slowly, with a long action time, increasing the feasibility of exogenous drug intervention. Therefore, targeted regulation of miRNAs has a very broad market prospect in sepsis-induced lung injury patients.

[0004] In current literature reports, miRNA-149-5p can inhibit ferroptosis in cardiomyocytes, thereby improving LPS-induced sepsis myocardial injury; it can also down-regulate endoplasmic reticulum stress in macrophages to improve acute lung injury caused by sepsis. However, there is little research on the effect of miRNA-149-5p on the function of sepsis endothelial cells, and its role is still unclear. Summary of the Invention

[0005] The present invention is directed to the treatment of sepsis-induced lung injury, aiming to provide a new therapeutic target for sepsis-induced lung injury and also to provide a new medical use of miRNA-149-5p. Specifically, it provides the application of miRNA-149-5p or its agonist in the preparation of drugs for the treatment of sepsis-induced lung injury, which can improve the function of vascular endothelial cells in sepsis and improve sepsis-induced lung injury.

[0006] The research process of the present invention is as follows: First, a sepsis endothelial cell model was established and sequenced to screen for differential microRNAs, and the expression of miRNA-149-5p was significantly downregulated; then, an miRNA-149-5p agonist (miRNA-149-5p agomir) was synthesized using it as a target, and cell experiments and animal model experiments were carried out. In vitro cell experiments showed that treating vascular endothelial cells with the miRNA-149-5p agonist could rescue the function of vascular endothelial cells under septic conditions, including improving their barrier function, reducing permeability; improving proliferation function and reducing their inflammatory level and reducing monocyte adhesion, etc.; in vivo animal experiments showed that treating with the miRNA-149-5p agonist could improve the pathological structure of sepsis-induced lung injury, reduce pulmonary edema and tissue protein exudation, and improve the pulmonary vascular barrier ability.

[0007] Based on the above research, the specific technical solution of the present invention is as follows:

[0008] In the first aspect of the present invention, there is provided the application of miRNA-149-5p or its agonist in the preparation of drugs for the treatment of sepsis-induced lung injury.

[0009] Preferably, sepsis-induced lung injury refers to acute lung injury occurring under septic conditions;

[0010] Preferably, the nucleotide sequence of miRNA-149-5p is as follows:

[0011] 3’-CCCUCACUUCUGUGCCUCGGUCU-5’ (SEQ ID NO.1);

[0012] The miRNA-149-5p agonist is miRNA-149-5p agomir, which is a double-stranded RNA molecule chemically synthesized based on the miRNA-149-5p sequence and capable of mimicking the function of endogenous miRNA-149-5p.

[0013] In the second aspect of the present invention, there is provided a recombinant expression vector of miRNA-149-5p, including an expression vector and miRNA-149-5p inserted and arranged on the expression vector, and the nucleic acid sequence of miRNA-149-5p is as shown in SEQ ID NO.1.

[0014] Among them, the expression vector is a conventional vector such as a plasmid vector, a cosmid vector, a phage vector or a viral vector, and the specific type is selected from the prior art according to the actual situation. The "viral vector" includes adeno-associated virus and lentivirus. Suitable viral vectors are well known to those of ordinary skill in the art. The remaining "non-viral vectors" include liposomes or lipid complexes, cationic polymers, chitosan polymers and nanoparticle vectors, etc. Suitable non-viral vectors are well known to those of ordinary skill in the art.

[0015] Correspondingly, the present invention also provides the use of the miRNA-149-5p recombinant expression vector in the preparation of a therapeutic drug for sepsis-induced lung injury.

[0016] The cell experiment results of the present invention found that the miRNA-149-5p agonist can reduce the expression of endothelial cell inflammatory factors IL-1β, IL-6 and adhesion molecule ICAM1, and at the same time up-regulate the expression of intercellular junction proteins Occludin, ZO-1 and VE-cadherin; it can reduce the adhesion between mononuclear endothelial cells, improve cell permeability, and improve the proliferation ability of endothelial cells.

[0017] The animal model experiment results showed that after treatment with the miRNA-149-5p agonist, the alveolar structure of the lung tissue of sepsis-induced lung injury mice was clearer and more complete, the alveolar septum was thinner, the pulmonary edema and the barrier function of pulmonary blood vessels were improved, and the protein exudation in the lung tissue was reduced.

[0018] In the third aspect of the present invention, a pharmaceutical composition for the treatment of sepsis-induced lung injury is provided, which includes an active component and a pharmaceutically acceptable excipient, and the active component includes miRNA-149-5p, an agonist of miRNA-149-5p or an overexpression vector of miRNA-149-5p.

[0019] In terms of the drug form, a drug or a pharmaceutical composition suitable for administration by a method selected from the following group: oral administration, injection (such as direct naked DNA or protein injection method, liposome-encapsulated DNA or mRNA injection method), gold-coated gene gun bombardment method, replication-defective adenovirus carrying the target DNA method or the protein encoded by the target gene, transdermal administration.

[0020] Generally, the liquid preparation can be stored stably for at least one year at 2°C - 8°C, and the lyophilized preparation remains stable at 30°C for at least six months. The preparation can be a suspension, aqueous injection, lyophilized preparation, etc. commonly used in the pharmaceutical field.

[0021] When the composition in the present invention is administered to animals including humans, the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the administration route, and can refer to the results of animal experiments and various situations, and the total dosage cannot exceed a certain range.

[0022] Furthermore, the pharmaceutical composition of the present invention can be used in combination with other drugs for sepsis-induced lung injury.

[0023] Functions and effects of the invention

[0024] The present invention proposes the potential therapeutic role of miRNA-149-5p in sepsis-induced lung injury. The results show that treatment of vascular endothelial cells with miRNA-149-5p agonists can rescue the functions of vascular endothelial cells under septic conditions, including improving their barrier function, reducing permeability, improving proliferation function, reducing their inflammatory level, and reducing monocyte adhesion, etc. It can also improve the pathological structure of the animal model of sepsis-induced lung injury, reduce pulmonary edema and tissue protein exudation, and improve the pulmonary vascular barrier ability. Therefore, the present invention proves the effect of miRNA-149-5p in the preparation of sepsis-induced lung injury, providing a new basis for the treatment of sepsis-induced lung injury. Description of the drawings

[0025] Figure 1 Shows the experimental results of miRNA-149-5p agonists improving the functions of vascular endothelial cells in sepsis. Among them, the results in Figure A show that compared with the control group, the expression of miRNA-149-5p in endothelial cells was significantly increased after intervention with miRNA-149-5p agonists; Figures B-C show that intervention with miRNA-149-5p agonists can reduce the expression of inflammatory factors IL-1β, IL-6 and adhesion molecule ICAM1 in endothelial cells, while upregulating the expression of intercellular junction proteins Occludin, ZO-1 and VE-cadherin; The results in Figure D confirm that intervention with miRNA-149-5p agonists can reduce the adhesion between mononuclear endothelial cells; Figure E shows that miRNA-149-5p agonists can improve the barrier function of endothelial cells and improve cell permeability; Figure F confirms that miRNA-149-5p agonists can improve the proliferation ability of endothelial cells.

[0026] Figure 2 Shows the experimental results of miRNA-149-5p agonists improving sepsis-induced lung injury: A: Compared with the control group, the alveolar structure of the lung tissue of mice in the miRNA-149-5p agonist intervention group is clearer and more complete, and the alveolar septum is thinner; B: The expression of miRNA-149-5p in the lung tissue increased after treatment with miRNA-149-5p agonists; C: The barrier function of pulmonary blood vessels in the miRNA-149-5p agonist intervention group was improved; D: The protein exudation in the lung tissue of the miRNA-149-5p agonist intervention group was reduced; E: The wet / dry ratio of the lung in the miRNA-149-5p agonist intervention group decreased, indicating improved pulmonary edema. Detailed implementation manners

[0027] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of traditional methods, such as the PCR method. Such methods are well known to those of ordinary skill in the art and have been described in many publications.

[0028] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described may be applied to the present invention. The preferred methods and materials described in the specific implementation manners are for illustrative purposes only.

[0029] The present invention discloses the role of miRNA-149-5p in the preparation of drugs for the treatment of sepsis-induced lung injury, and in vitro cell experiments and in vivo animal experiments are carried out respectively, as follows:

[0030] I. Cell experiments

[0031] The cell experiments used human umbilical vein endothelial cells (HUVECs) as the research object, and an in vitro sepsis endothelial cell model was constructed by LPS stimulation to explore the effect of miRNA-149-5p on the function of in vitro sepsis vascular endothelial cells. The cell experiment groups were as follows: control group (NC + LPS), miRNA-149-5p agonist intervention group (miRNA-149-5p mimic + LPS). Among them, a miRNA-149-5p agonist with a final concentration of 50 nM was added to the culture medium of the cells in the miRNA-149-5p agonist group, and an agonist control with a final concentration of 50 nM was added to the control group. 48 hours after administration, the cells in each group were stimulated with LPS to establish a sepsis endothelial cell model, and functional experiments were performed on the cells 24 hours later. PCR and WB were used to detect the expression of endothelial cell inflammatory factors, adhesion molecules, and intercellular junction proteins; a mononuclear endothelial cell adhesion experiment was used to detect the adhesion ability of endothelial cells; a permeability experiment was used to detect the barrier function of endothelial cells, and a CCK-8 experiment was used to detect the proliferation ability of endothelial cells.

[0032] The results are as Figure 1 shown, which show the experimental results of the improvement of sepsis vascular endothelial cell function by the miRNA-149-5p agonist. 48 hours before the construction of the cell sepsis model, the vascular endothelial cells were treated with the miRNA-149-5p agonist or negative control. Compared with the control group, the expression of miRNA-149-5p in endothelial cells was significantly increased after miRNA-149-5p agonist intervention ( Figure 1A); Intervention with miRNA-149-5p agonist can reduce the expression of endothelial cell inflammatory factors IL-1β, IL-6 and adhesion molecule ICAM1, while up-regulating the expression of intercellular junction proteins Occludin, ZO-1 and VE-cadherin ( Figure 1 B-C); Intervention with miRNA-149-5p agonist can reduce the adhesion between monocytes and endothelial cells ( Figure 1 D); miRNA-149-5p agonist can improve the barrier function of endothelial cells and improve cell permeability ( Figure 1 E); miRNA-149-5p agonist can improve the proliferation ability of endothelial cells ( Figure 1 F).

[0033] In summary, compared with the control group, increasing the expression level of miRNA-149-5p in septic vascular endothelial cells can significantly reduce the inflammatory level of cells, reduce the adhesion of monocytes, and improve the barrier function and proliferation ability of cells.

[0034] II. Animal experiments

[0035] The animal experiment used C57 wild-type mice as the research object, and simulated human sepsis by cecal ligation and puncture (CLP). After CLP modeling, the mice were randomly divided into the following two groups: control group (agomir NC), miRNA-149-5p agonist intervention group (miRNA-149-5p agomir). The miRNA-149-5p agonist dry powder was dissolved in normal saline, and according to the dosing dose in previous literature, the mice were immediately instilled with miRNA-149-5p agonist or NC 5 nmol through the airway after CLP modeling. After 48 hours, the lung tissues of the mice were collected for paraformaldehyde fixation for HE staining, wet / dry ratio detection, Evans blue detection and total alveolar protein detection.

[0036] Figure 2 The experimental results showing that miRNA-149-5p improves septic lung injury are presented. The results show that compared with the control group, the alveolar structure of the lung tissue of the mice in the miRNA-149-5p agonist intervention group is clearer and more complete, and the alveolar septum is thinner ( Figure 2 A); The expression of miRNA-149-5p in the lung tissue is increased after miRNA-149-5p agonist treatment ( Figure 2 B); The barrier function of the pulmonary blood vessels in the miRNA-149-5p agonist intervention group is improved ( Figure 2 C); The protein exudation in the lung tissue of the miRNA-149-5p agonist intervention group is reduced ( Figure 2 D); The wet / dry ratio of the lung in the miRNA-149-5p agonist intervention group is decreased, indicating improved pulmonary edema (Figure 2 E).

[0037] Animal experiment results showed that compared with the control group, administration of the miRNA-149-5p agonist could improve the pathological structure of the lung tissue in sepsis, reduce pulmonary edema and protein exudation, and improve the pulmonary vascular barrier function.

[0038] In summary, treatment with the miRNA-149-5p agonist can alleviate sepsis-induced lung injury, and it has good efficacy and promising prospects for the preparation of therapeutic drugs for sepsis-induced lung injury.

[0039] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, various changes and improvements will occur to the present invention, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

Use of miRNA-149-5p or its agonist in the preparation of a pharmaceutical composition for treating septic lung injury.

2. The application according to claim 1, wherein The septic lung injury is acute lung injury occurring under septic conditions.

3. The application according to claim 1, characterized in that, The nucleic acid sequence of the miRNA-149-5p is as shown in SEQ ID NO.1; the miRNA-149-5p agonist is miRNA-149-5p mimic or agomir, which is a chemically synthesized double-stranded RNA molecule that can mimic the function of endogenous miRNA-149-5p.

4. The application according to claim 1, characterized in that The drug is a drug for improving the function of vascular endothelial cells under septic conditions or improving the pathological conditions of septic acute lung injury.

5. The application according to claim 4, wherein The drug for improving the function of vascular endothelial cells under septic conditions is a drug for improving endothelial cell barrier function, promoting cell proliferation, reducing monocyte adhesion or reducing the inflammatory level of cells; the drug for improving the pathological conditions of septic acute lung injury includes drugs for improving the pathological structure of lung tissue, reducing pulmonary edema and tissue protein exudation or improving the pulmonary vascular barrier ability.

6. A recombinant expression vector of miRNA-149-5p, characterized in that, It includes an expression vector and miRNA-149-5p inserted and set on the expression vector, and the nucleic acid sequence of miRNA-149-5p is as shown in SEQ ID NO.

1.

7. Use of the miRNA-149-5p recombinant expression vector according to claim 6 in the preparation of a drug for treating septic lung injury.

8. A pharmaceutical composition for treating sepsis-induced lung injury, characterized in that, It includes an active ingredient and a pharmaceutically acceptable excipient, and the active ingredient includes the miRNA-149-5p or its agonist according to any one of claims 1 to 3, or the recombinant expression vector of miRNA-149-5p according to claim 6.

9. The pharmaceutical composition for treating sepsis-induced lung injury according to claim 8, wherein The preparation form of the pharmaceutical composition is any one of oral preparations, injections, and drops.

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