Application of miR-23b in preparation of medicine for treating EndoMT related diseases

Abstract

The invention relates to the technical field of medicines, and provides application of miR-23b in preparation of a medicine for treating EndoMT related diseases. Experimental results show that the miR-23b can significantly inhibit the expression level of SMAD3 and downstream effector protein thereof, further inhibit the expression of EndoMT and inflammatory cell adhesion / inflammatory response related genes, increase the expression of cell barrier protein related genes, and improve the cell proliferation activity at the same time; the traditional Chinese medicine composition has a positive effect on treatment of endothelial cell dysfunction in sepsis. Therefore, the invention provides a new basis for the miR-23b 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 miR-23b in the preparation of drugs for treating EndoMT-related diseases. Background Art

[0002] According to the latest international consensus, sepsis is defined as a life-threatening organ dysfunction caused by an uncontrolled host response to infection. It remains a major threat to global health. In 2017, approximately 48.9 million cases and 11 million sepsis-related deaths were reported worldwide, accounting for approximately 20% of global deaths annually. Sepsis can cause systemic reactions and damage to various organs, such as sepsis-related encephalopathy, acute lung injury (ALI), sepsis-related acute kidney injury, and disseminated intravascular coagulation (DIC). A key common feature of these diseases is endothelial cell (EC) damage and dysfunction. The physiological functions of endothelial cells include regulating coagulation, releasing growth factors, and regulating blood flow through the formation, contraction, and relaxation of selective barriers. When endothelial cells are damaged, they can lead to tissue edema and widespread inflammation. According to epidemiological surveys, the most common site of infection in sepsis is the lungs (68.2%), primarily related to endothelial barrier dysfunction leading to increased vascular permeability and increased transendothelial migration of leukocytes, resulting in an excessive inflammatory response. Therefore, endothelial cell dysfunction is one of the main culprits mediating septic lung injury.

[0003] In recent years, the contemporary definition of endothelial dysfunction has expanded to encompass a range of cellular events, including oxidative stress, inflammation / leukocyte adhesion, endothelial-mesenchymal transition (EndoMT), mitochondrial dysfunction, aging, and dysregulated endothelial cell metabolism. EndoMT represents the process by which endothelial cells disaggregate, change their shape into mesenchymal-like cells, and migrate into surrounding tissues, an example of which is the development of heart valves. EndoMT is regulated by multiple signaling pathways, including TGF-β and BMP signaling, and can transform and differentiate endothelial cells into myofibroblasts. Overexpression in pathological conditions, such as in atherosclerosis, hypertension, diabetes, cancer, and infection, contributes to endothelial dysfunction and vascular fibrosis. EndoMT can be activated by the TGF-β1 / SMAD3 signaling pathway, and thus, it can be inhibited by targeting SMAD3 or TGF-β1.

[0004] MicroRNAs (miRNAs) are a class of small, noncoding RNA molecules typically around 22 nt in length. Their primary function is to bind to target mRNAs and form miRNA-induced silencing complexes, silencing transcripts or preventing their translation. Although microRNAs were first discovered in 2001 and are numerous, their role in sepsis-induced lung injury is still in its early stages. Therefore, the role of miRNAs in vascular endothelial cells in septic lung injury may hold broad promise. SMAD3 primarily participates in the TGF-β1-mediated signaling pathway, transmitting signals to the nucleus and playing multiple regulatory roles in sepsis. Studies have shown that the TGF-β1 / SMAD3 signaling pathway can inhibit the production of proinflammatory cytokines by monocytes and macrophages, but it also activates the glycolytic pathway in monocytes and macrophages, promoting coagulation and leading to reduced survival in septic mice. Furthermore, the TGF-β1 / SMAD3 signaling pathway has been shown to activate pyroptosis in animal models of septic renal injury.

[0005] Traditional pathways for inhibiting EndoMT primarily include the NF-κb pathway or Toll-like receptors, but both only indirectly affect endothelial cell function by reducing inflammation. Secondly, compared to traditional mRNA gene knockout or siRNA interference, inhibiting target mRNA through miRNA is more consistent with natural physiological mechanisms and is more stable within cells. The regulation of mRNA is more precise and reversible, and their effects on non-target mRNAs are often smaller, which can better avoid off-target effects. Because miRNA regulatory mechanisms involve incomplete complementary pairing, they can better tolerate slight sequence variations, which helps reduce off-target issues caused by single nucleotide polymorphisms (SNPs). Therefore, there is a need for an effective and direct miRNA that can restore endothelial cell morphology and function while altering the inflammatory state, as well as downstream target pathways, to treat dysfunction in sepsis endothelial cell models. Summary of the Invention

[0006] The present invention is directed to the above-mentioned problems, and aims to provide a new therapeutic target for septic lung injury, a new target for inhibiting EndoMT, and a new medical use of miR-23b; specifically, it provides the use of miR-23b in the preparation of therapeutic drugs for EndoMT-related diseases including septic lung injury, which can improve the function of vascular endothelial cells in sepsis and improve septic lung injury.

[0007] The research process of this invention is as follows: Initially, high-throughput sequencing and bioinformatics analysis, as well as related experiments, were performed on serum samples collected dynamically from 10 patients with extensive burns (>80% body surface area) in an explosion accident. Comparison with public databases revealed that has-miR-23b-3p (miR-23b) was strongly associated with SMAD3 and may play a key role in regulating systemic inflammation and immune homeostasis. This gene was significantly downregulated in patients with sepsis. miR-23b directly targets and regulates SMAD3, significantly inhibiting the expression of SMAD3 and its downstream effector proteins. This in turn inhibits the expression of genes related to EndoMT and inflammatory cell adhesion / inflammatory responses, increases the expression of genes related to cell barrier proteins, and enhances cell proliferation. This has a positive effect on the treatment of endothelial cell dysfunction in sepsis. EndoMT is activated by the TGF-β1 / SMAD3 signaling pathway, and miR-23b may inhibit EndoMT by targeting SMAD3.

[0008] Based on the above research, the specific technical solutions of the present invention are as follows:

[0009] The first aspect of the present invention provides the use of miR-23b in the preparation of a drug for treating EndoMT-related diseases.

[0010] Preferred EndoMT-related diseases include fibrotic diseases (e.g., cardiac fibrosis, pulmonary fibrosis, and renal fibrosis), vascular diseases (atherosclerosis, pulmonary arterial hypertension (PAH)), and cancer-related fibrosis (breast cancer, pancreatic cancer). The present invention primarily relates to septic lung injury, which also involves pulmonary fibrosis. Therefore, the present invention also provides the use of miR-23b in the preparation of a therapeutic drug for treating septic lung injury.

[0011] Furthermore, septic lung injury refers to acute lung injury occurring under septic conditions.

[0012] Furthermore, the drug is a drug that directly targets and regulates SMAD3 and thereby inhibits EndoMT.

[0013] In the present invention, miR-23b is selected from any one of the following situations:

[0014] (1) Liposomes or nanocarriers encapsulating miR-23b;

[0015] (2) miR-23b recombinant expression vector;

[0016] (3) miR-23b mimic, the nucleotide sequence of miR-23b or its mimic is as follows:

[0017] 5'-AUCACAUUGCCAGGGAUUACCAC-3' (SEQ ID NO. 1).

[0018] In a second aspect, the present invention provides a miR-23b recombinant expression vector, comprising an expression vector and miR-23b inserted into the expression vector.

[0019] The expression vector is a conventional vector such as a plasmid vector, cosmid vector, phage vector, or viral vector. The specific type is selected from the prior art based on actual conditions. The "viral vector" includes adeno-associated virus and lentivirus. Suitable viral vectors are well known to those of ordinary skill in the art. Other "non-viral vectors" include liposomes or lipid complexes, cationic polymers, chitosan polymers, and nanoparticle vectors. Suitable non-viral vectors are well known to those of ordinary skill in the art.

[0020] Correspondingly, the present invention also provides the use of a miR-23b recombinant expression vector in the preparation of a drug for treating septic lung injury.

[0021] By transfecting septic endothelial cells with miR-23b mimic, it was found that in the septic endothelial cell model after treatment, the miR-23b content increased, the inflammation decreased, the expression of adhesion molecules decreased, and the expression of barrier proteins increased; at the same time, the expression of the TGF-β1 / SMAD3 signaling pathway decreased, and the EndoMT situation improved; the permeability and adhesion of the model were improved, and the cell proliferation activity increased.

[0022] In a third aspect, the present invention provides a pharmaceutical composition for treating EndoMT-related diseases (such as septic lung injury), comprising an active ingredient and a pharmaceutically acceptable excipient. The active ingredient includes a liposome or nanocarrier encapsulating miR-23b, a miR-23b mimic, or a miR-23b overexpression vector.

[0023] In terms of drug form, the drug or pharmaceutical composition is suitable for administration by a method selected from the following groups: oral administration, injection (such as direct naked DNA or protein injection, liposome-encapsulated DNA or mRNA injection), gold-coated gene gun bombardment, replication-defective adenovirus carrying target DNA or protein encoded by the target gene, and transdermal administration.

[0024] Typically, liquid preparations are stable for at least one year at 2°C-8°C, and lyophilized preparations are stable for at least six months at 30°C. These preparations can be commonly used in the pharmaceutical field, such as suspensions, aqueous injections, and lyophilized preparations.

[0025] When the composition of the present invention is administered to animals including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and administration route. The results of animal experiments and various circumstances can be referred to, and the total dosage cannot exceed a certain range.

[0026] Furthermore, the pharmaceutical composition of the present invention can be used in combination with other drugs for septic lung injury.

[0027] Functions and effects of the invention

[0028] The present invention proposes the potential therapeutic effect of miR-23b in septic lung injury. The present invention uses miR-23b as a gene regulation tool, which is closer to the natural physiological mechanism and more stable in the cell; the regulation of mRNA is more precise and reversible, and their impact on non-target mRNA is often smaller, which can better avoid off-target effects. By inhibiting the SMAD3 signaling pathway, miR-23b can be delivered into the recipient cells, significantly increasing the content of miR-23b in the cells, thereby inhibiting SMAD3 transcriptional expression, reducing EndoMT in the sepsis endothelial cell model, reducing inflammatory factor gene expression, and restoring cell barrier function and proliferation activity. Therefore, the present invention demonstrates the effect of miR-23b in the preparation of septic lung injury and provides a new basis for the treatment of septic lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure 3: Inflammatory response and dysfunction in the sepsis endothelial cell model: (A) Dual luciferase reporter assay results; (B) Expression levels of inflammatory factors under different LPS concentrations, with Ctrl as the control group and the rest as LPS groups, verifying the successful construction of the sepsis endothelial cell model; (C) Western blot results of barrier protein expression in the sepsis endothelial cell model; (D) Permeation assay in the sepsis endothelial cell model; (E) Monocyte adhesion assay in the sepsis endothelial cell model; (F) CCK8 proliferation assay in the sepsis endothelial cell model.

[0030] Figure 2Figures show the expression of miR-23b, TGF-β1 / SMAD3 pathway, and EndoMT markers in the sepsis endothelial cell model: (A) Real-time quantitative PCR results of miR-23b transcription level; (B) Real-time quantitative PCR results of TGF-β1 / SMAD3 pathway transcription level; (C) Western blot results of TGF-β1 / SMAD3 pathway translation; (D) Real-time quantitative PCR results of EndoMT marker transcription level, where CDH2 and VIM are mesenchymal cell markers, and vWF and CD31 are endothelial cell markers; (E) Western blot results of EndoMT marker translation.

[0031] Figure 3 Figures show the improvement in molecular protein levels in the sepsis endothelial cell model after miR-23b treatment: (A) miR-23b transcription level, where Vector Control is the positive control; (B) Real-time quantitative PCR results for inflammatory factor transcription levels after treatment; (C) Real-time quantitative PCR results for adhesion molecule and barrier protein transcription levels after treatment, where ICMA1 is an adhesion molecule and TJP1, OCLN, and CDH5 are barrier proteins; (D) Western blot results for the translation of adhesion molecules and barrier proteins after treatment; (E) Real-time quantitative PCR results for the transcription level of the TGF-β1 / SMAD3 pathway after treatment; (F) Western blot results for the translation of the TGF-β1 / SMAD3 pathway after treatment; (G) Real-time quantitative PCR results for the transcription level of EndoMT markers after treatment; (H) Western blot results for the translation of EndoMT markers after treatment.

[0032] Figure 4 Figures show the improvement in the functional phenotype of the septic endothelial cell model after treatment with miR-23b: (A) After treatment, the results of cell immunofluorescence staining are shown, where green indicates adhesion molecule proteins and barrier proteins, and blue DAPI indicates cell nuclei; (B) After treatment, the results of the monocyte adhesion assay are shown, where blue indicates monocytes; (C) After treatment, the results of the fluorescence detection assay of the endothelial cell permeability assay are shown; (D) After treatment, the results of the CCK8 cell proliferation activity assay are shown in the septic endothelial cell model. DETAILED DESCRIPTION

[0033] 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 detailed descriptions of conventional methods, such as PCR. Such methods are well known to those skilled in the art and are described in numerous publications.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0035] This study identifies a novel approach to inhibiting endothelial cell dysfunction in sepsis models by targeting the SMAD3 pathway via miR-23b, and provides methods for preparing and using this miRNA. miR-23b significantly inhibits the expression of SMAD3 and its downstream effector proteins, thereby suppressing the expression of genes associated with EndoMT and inflammatory cell adhesion / inflammatory response, while increasing the expression of genes associated with cell barrier proteins and enhancing cell proliferation. This approach has a positive effect on the treatment of endothelial cell dysfunction in sepsis.

[0036] To achieve the above objectives, the present invention provides a new approach that targets SMAD3 through miR-23b to inhibit dysfunction in a sepsis endothelial cell model; wherein the miR-23b includes a complete coding gene sequence.

[0037] Preferably, this approach targets SMAD3 through miR-23b to inhibit dysfunction in the sepsis endothelial cell model. By transfection with lipofectamine 2000, miR-23b can be delivered into the recipient cells, significantly increasing the intracellular miR-23b content, thereby inhibiting SMAD3 transcriptional expression, reducing EndoMT in the sepsis endothelial cell model, reducing inflammatory factor gene expression, and restoring cell barrier function and proliferation activity.

[0038] Furthermore, in the above approach, the sepsis endothelial cell model is a HUVEC cell line. The sepsis endothelial cell model is prepared by the following method:

[0039] A. HUVEC cell line culture: HUVECs were cultured in a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (high-glucose DMEM: Ham's F12 medium = 3:1) at 37°C and 5% CO2 to a cell density of 60%-70%.

[0040] B. Lipopolysaccharide (LPS) effect: LPS was added to the cell culture medium to a final concentration of 10 μg / mL and the effect lasted for 24 h.

[0041] The HUVEC cell line prepared by the above method is considered to be a sepsis cell model, in which the expression of inflammatory factors is increased, endothelial cell function is disordered, and proliferation activity is decreased.

[0042] Furthermore, in the above approach, the lipofectamine 2000 transfection comprises the following steps:

[0043] 1. Dilute lipofectamine 2000 and serum-free culture medium at a volume ratio of 1:50 to form a mixture a;

[0044] 2. Mixture a was mixed with miR-23b mimic to form mixture b, wherein the volume ratio of lipofectamine 2000 and miR-23b mimic (concentration 20 μM) was 1:1;

[0045] 3. Add mixture b to the cell dish and culture the cells for 24h~96h.

[0046] The preparation method of miR-23b mimic in step 2 is pure chemical synthesis, and its sequence is 5'-AUCACAUUGCCAGGGAUUACCAC-3' (SEQ ID NO. 1).

[0047] The specific embodiments are as follows:

[0048] Example 1 Construction and identification of sepsis endothelial cell model

[0049] 1. Cell preparation: HUVEC cells were revived and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (DMEM medium for short) at 37°C and 5% CO2 in a constant temperature incubator. After 1-2 passages, they were inoculated into different cell culture dishes. For example, a total of 6×10 5 cells.

[0050] 2. Construction of a sepsis endothelial cell model: When the cell density reaches 60-70%, wash the cells in the six-well plate three times with PBS and add LPS working solution. Depending on the specifications of the culture dish, the final LPS concentration is controlled at 10 μg / mL.

[0051] 3. Validation of the Sepsis Endothelial Cell Model: After 24 hours of LPS exposure, samples were collected. Real-time quantitative PCR, Western blot, monocyte adhesion assay, and CCK8 cell proliferation assay were used to examine the expression of inflammatory factors, adhesion molecules, and barrier proteins in HUVECs, as well as their functional phenotypes. The details are as follows:

[0052] ① Cell proliferation experiment: cells were seeded in 96-well plates, with 5×10 cells per well. 3 After treatment according to experimental groups, the absorbance OD values ​​were measured using CCK-8 kit after culturing for 0 h, 24 h, 48 h, and 72 h.

[0053] ② Real-time fluorescence quantitative PCR: Total RNA from cells and tissues was isolated and extracted using Trizol reagent, and then real-time quantitative PCR analysis was performed using SYBR Green PCR Master Mix (total volume of 10 ml) and Step One Plus real-time PCR System (Applied Biosystems).

[0054] ③Westing Blot: Treat cells and tissues with Repa lysis buffer containing protease inhibitors. Centrifuge at 4°C, 12,000 rpm for 10 min, collect the supernatant for BSA protein quantification, and then load the sample for electrophoresis, transfer to a membrane, block, incubate with antibodies, and expose according to the experimental group. Image J is used to process the image and calculate the grayscale value.

[0055] ④Monocyte adhesion assay: HUVECs were seeded in a 24-well plate and cultured in a 37°C, 5% CO2 incubator until the cell confluence reached 100% to form a monolayer. Thp1 monocytes were stained with a live cell stain (Hoechst 33342) at 37°C for 3.30 min and then seeded on the monolayer of HUVECs. Approximately 1×10 5 The cells were cultured in a 37°C, 5% CO2 incubator for about 2 hours, and non-adherent monocytes were washed away with PBS. The adhesion of monocytes was observed under a fluorescence microscope.

[0056] ⑤ Endothelial cell permeation experiment: Cell digestion, counting and adjusting the cell concentration to 1×10 5 1 mL of cell suspension was pipetted into a 3 μm pore Transwell chamber, which was then inserted into a 24-well cell culture plate. 1.5 mL of complete cell culture medium was pre-filled into the lower chamber of the culture plate. The plate was placed in a 37°C, 5% CO2 incubator. After reaching 100% confluency and forming a stable monolayer, 1.5 mL of FITC-Dextran reagent was added to the upper chamber and incubated at room temperature in the dark for 20 minutes. The lower chamber solution was aspirated into a 96-well plate, with 100 μL added to each well, in triplicate. The absorbance at 485 / 535 nm was measured using a multi-functional microplate reader. Data were collected, normalized, and statistically analyzed.

[0057] The experimental results are as follows Figure 1 and Figure 2 As shown: The sepsis endothelial cell model was successfully established, in which 10 μg / mL was able to significantly increase the expression of inflammatory factors. Figure 1 (A); At the same time, the expression of adhesion molecules increased and the expression of barrier proteins decreased, see Figure 1(BC); The permeation and adhesion of the model are more serious, and the cell proliferation activity is reduced. Figure 1 (DF). The expression of miR-23b decreased in the model, see Figure 2 (A); TGF-β1 / SMAD3 signaling pathway expression is activated, see Figure 2 (BC); EndoMT worsens, see Figure 2 (DE).

[0058] Example 2 miR-23b mimic transfection

[0059] 1. Cell preparation: The specific operation is the same as Example 1.

[0060] 2. Construction of sepsis endothelial cell model: The specific operation is the same as Example 1.

[0061] 3. Preparation of miR-23b mimic: The designed sequence was 5'-AUCACAUUGCCAGGGAUUACCAC-3', and it was synthesized on a pure chemical automated DNA / RNA synthesizer for solid phase synthesis and purified by polyacrylamide gel electrophoresis.

[0062] 4. Transfection of miR-23b mimic: Prepare a mixture of lipofectamine 2000 and serum-free medium (volume ratio: 1:50). Mix mixture a with miR-23b mimic to form mixture b, where the volume ratio of lipofectamine 2000 to miR-23b mimic (20 μM) is 1:1. Add mixture b to cells and culture for 24–96 hours.

[0063] 5. Verification of miR-23b, inflammation, adhesion molecules, and barrier protein molecular levels after treatment: The specific operation is the same as step 3 of Example 1.

[0064] 6. Functional phenotype detection of the model after treatment: The specific operation is the same as step 3 of Example 1.

[0065] The experimental results can be found in Figure 3 and Figure 4 : In the sepsis endothelial cell model after treatment, miR-23b content increased, see Figure 3 (A); Inflammation is reduced, see Figure 3 (B); At the same time, the expression of adhesion molecules decreased and the expression of barrier proteins increased, see Figure 3 (CD) and Figure 4 (A); TGF-β1 / SMAD3 signaling pathway expression decreased, see Figure 3(EF); EndoMT has improved, see Figure 3 (GH); The permeability and adhesion of the model were improved, and the cell proliferation activity was increased. Figure 4 (BD).

[0066] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of miR-23b in the preparation of drugs for the treatment of EndoMT-related diseases.

2. Application of miR-23b in the preparation of drugs for the treatment of septic lung injury.

3. The use according to claim 2, characterized in that The septic lung injury is acute lung injury occurring under sepsis conditions.

4. The use according to claim 1 or 2, characterized in that The drug is a drug that directly targets and regulates SMAD3 and thereby inhibits EndoMT.

5. The use according to claim 1, characterized in that The miR-23b is selected from any one of the following situations: (1) Liposomes or nanocarriers encapsulating miR-23b; (2) miR-23b recombinant expression vector; (3) miR-23b mimics.

6. The use according to claim 5, characterized in that The nucleotide sequence of the miR-23b or miR-23b mimic is shown in SEQ ID NO.

1.

7. A miR-23b recombinant expression vector, characterized in that: The invention comprises an expression vector and miR-23b inserted into the expression vector. The nucleic acid sequence of miR-23b is shown as SEQ ID NO.

1.

8. Use of the miR-23b recombinant expression vector according to claim 7 in the preparation of a drug for treating septic lung injury.

9. A pharmaceutical composition for treating septic lung injury, characterized in that: It comprises an active component and a pharmaceutically acceptable excipient, wherein the active component comprises the miR-23b according to claim 5.

10. The pharmaceutical composition for treating septic lung injury according to claim 9, characterized in that: The pharmaceutical composition is in the form of an oral preparation, an injection, or a drop.

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