Application of miR-144 inhibitor in preparation of medicine for enhancing curative effect of glucocorticoid on treating ARDS and composition of miR-144 inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and relates to application of miRNA targeted regulation in treatment of acute respiratory distress syndrome, in particular to application of a miR-144 inhibitor in preparation of a medicine for improving glucocorticoid treatment sensitivity of an ARDS patient. Research on a lipopolysaccharide-induced ARDS rat model finds that miR-144 participates in regulation and control of glucocorticoid receptor beta (GR beta) and NF-kappa B signal pathways in the ARDS inflammatory reaction process, the number of neutrophils in alveolar lavage fluid and the level of inflammatory factors such as IL-6 and TNF-alpha can be remarkably reduced by using the miR-144 inhibitor and glucocorticoid for treatment, and the application of the miR-144 inhibitor to treatment of the pulmonary alveolar lavage fluid in the treatment of the pulmonary alveolar lavage fluid is broad. The wet-to-dry ratio and pathological injury score of lung tissues are reduced, and GR beta and NF-kappa B expression is inhibited, so that the anti-inflammatory effect of glucocorticoid is remarkably enhanced. The invention provides a treatment strategy for improving glucocorticoid resistance of ARDS patients by taking miR-144 as a target for the first time, and provides a new drug target and a technical scheme for accurate treatment of ARDS.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to the application of miRNA targeted regulation technology in the treatment of acute respiratory distress syndrome, specifically the application of miR-144 inhibitors as glucocorticoid sensitizers in the preparation of drugs for treating ARDS. Background Technology

[0002] Acute respiratory distress syndrome (ARDS) is a common type of acute respiratory failure in clinical critical care medicine, characterized by progressive hypoxemia, diffuse alveolar damage, and uncontrolled pulmonary inflammation. Optimizing ARDS treatment is a pressing medical challenge that needs to be addressed.

[0003] The core pathophysiology of ARDS is non-cardiac pulmonary edema caused by increased permeability of alveolar epithelium and capillary endothelium. Essentially, it stems from an uncontrolled inflammatory cascade within the lungs, with neutrophil infiltration and the massive release of pro-inflammatory factors (such as IL-6 and TNF-α) being key factors exacerbating lung injury. Glucocorticoids (GCs), as classic and potent anti-inflammatory drugs, can alleviate lung injury through mechanisms such as reducing vascular permeability and inhibiting the release of inflammatory factors. However, their clinical efficacy exhibits significant heterogeneity, with some patients showing marked GC resistance, severely limiting their therapeutic value.

[0004] The anti-inflammatory effect of glucocorticoids (GCs) is mainly achieved by binding to glucocorticoid receptor α (GRα), while glucocorticoid receptor β (GRβ) competitively antagonizes GRα function and is a core molecule leading to GC resistance. Previous studies have confirmed that GRβ expression is significantly elevated in GC-resistant patients, but the regulatory mechanism of GRβ expression in ARDS remains unclear.

[0005] On the other hand, microRNAs (miRNAs) are a class of small non-coding RNAs that act as post-transcriptional regulatory molecules, regulating gene expression by targeting and binding to the 3' untranslated region (UTR) of target genes. Studies have shown that miR-144 can target and bind to the 3'UTR of the GRβ gene and regulate its expression. Existing research indicates that miR-144 can participate in the regulatory processes of various diseases. For example, Chinese patent CN104548134A discloses the application of miR-144 and its inhibitors in myocardial oxidative stress injury, revealing that miR-144 can inhibit Nrf2 gene expression and promote myocardial oxidative stress. In bladder cancer cells, using the peptide nucleic acid molecule "Sweet-P" to block the binding of miR-144 to the GRβ 3'UTR can inhibit cancer cell migration. However, the role and mechanism of miR-144 in ARDS GC resistance have not been reported, and there is a lack of targeted intervention strategies. Furthermore, there is no existing documentation or inspiration regarding the application of miR-144 inhibitors to enhance the therapeutic effect of ARDS GC.

[0006] Therefore, developing interventions targeting miR-144 to improve the sensitivity of ARDS patients to GC treatment has significant clinical implications and application value. Summary of the Invention

[0007] The purpose of this invention is to provide the application of miR-144 inhibitors as glucocorticoid sensitizers in the preparation of drugs for the treatment of ARDS. The aim is to clarify the regulatory role and molecular mechanism of miR-144 in the sensitivity of ARDS patients to glucocorticoid therapy, and to provide a GC sensitization strategy targeting miR-144, so as to provide new drug targets and technical support for improving ARDS GC resistance and optimizing treatment regimens in clinical practice.

[0008] On the one hand, the present invention provides the use of miR-144 inhibitors in the preparation of drugs that enhance the efficacy of glucocorticoid therapy for acute respiratory distress syndrome.

[0009] Preferably, the glucocorticoid is one or more of methylprednisolone sodium succinate, dexamethasone, or hydrocortisone.

[0010] Preferably, the miR-144 inhibitor includes one or more of nucleic acid inhibitors or small molecule inhibitors that specifically inhibit miR-144 expression.

[0011] Preferably, the nucleic acid inhibitor is one or more of the following: miR-144 antisense oligonucleotide, siRNA, shRNA, microRNA inhibitor, and nucleic acid aptamer.

[0012] Preferably, the drug is a combination of a miR-144 inhibitor and a glucocorticoid; the drug dosage form is one or more of an injection, a suspension, a lyophilized powder for injection, or a liposome preparation.

[0013] Preferably, in the composition, the dosage of the miR-144 inhibitor is 20-30 nmol / kg, and the dosage of the glucocorticoid is 1.5-2.5 mg / kg.

[0014] Preferably, the miR-144 inhibitor is administered at a dose of 25 nmol / kg, and the glucocorticoid is methylprednisolone sodium succinate, administered at a dose of 2 mg / kg.

[0015] Preferably, the drug exerts its sensitizing effect by downregulating GRβ and NF-κB expression, inhibiting pulmonary inflammatory response, and reducing pulmonary edema.

[0016] On the other hand, the present invention also provides a pharmaceutical composition for enhancing the efficacy of glucocorticoid therapy for acute respiratory distress syndrome, the pharmaceutical composition comprising an effective amount of a miR-144 inhibitor and a pharmaceutically acceptable carrier.

[0017] Preferably, the pharmaceutical composition further comprises a glucocorticoid.

[0018] By adopting the above technical solutions, the sensitivity of ARDS patients to GC treatment can be improved, providing new drug targets and technical support for clinical improvement of ARDS GC resistance and optimization of treatment plans.

[0019] Compared with the prior art, the beneficial effects of the present invention are: This invention reveals for the first time that miR-144 is a key molecule regulating glucocorticoid resistance in ARDS. miR-144 inhibitors can significantly enhance the anti-inflammatory effects of glucocorticoids and improve pulmonary edema and pathological damage to lung tissue. The mechanism is related to the downregulation of GRβ and NF-κB expression. Specifically: 1. This invention is the first to apply miR-144 inhibitors to the treatment of ARDS and to verify their synergistic effect with glucocorticoids.

[0020] 2. The molecular mechanism by which miR-144 inhibitors downregulate GRβ expression and reverse GC resistance has been clarified, providing a new strategy for treating GC-insensitive ARDS patients.

[0021] 3. This invention also provides specific dosage regimens for the combined use of miR-144 inhibitors and glucocorticoids. Combined treatment with glucocorticoids significantly reduces inflammation levels. By inhibiting miR-144 expression, it reduces the activity of GRβ and NF-κB signaling pathways, thereby improving the sensitivity of ARDS patients to glucocorticoid therapy and enhancing the efficacy of glucocorticoid therapy for ARDS, laying the foundation for clinical translation. Attached Figure Description

[0022] Figure 1 This is a flowchart of the experimental process of the present invention.

[0023] Figure 2 This is a graph showing the trend of body weight change in rats in each group according to the embodiments of the present invention. The horizontal axis D0 represents the period before intervention, and D1-D5 represent the number of days after intervention. § p < 0.05 §§ p < 0.01, compared with D0; ** p < 0.01, compared with the model group; # p < 0.05 ## p < 0.01, compared with the GC group; && p < 0.01, compared with the GC + miR-144 simulant group.

[0024] Figure 3 This is a comparison chart of neutrophil counts in BALF from different groups in the embodiments of the present invention. ** p < 0.01, compared with the model group; ## p < 0.01, compared with the GC group; && p < 0.01, compared with the GC + miR-144 simulant group.

[0025] Figure 4 This is a comparison chart of inflammatory factors in BALF from different groups in the embodiments of the present invention. Chart A shows the concentration of IL-6, and Chart B shows the concentration of TNF-α. ** p < 0.01, compared with the model group; ## p < 0.01, compared with the GC group; && p < 0.01, compared with the GC + miR-144 simulant group.

[0026] Figure 5 This is a comparison chart of the wet-to-dry ratio of lung tissue in various groups according to embodiments of the present invention. * p < 0.05 ** p < 0.01, compared with the model group; # p < 0.05, compared with the GC group; && p < 0.01, compared with the GC + miR-144 simulant group.

[0027] Figure 6 These are comparative images of lung tissue pathological sections and pathological scores from various groups in the embodiments of the present invention. * p < 0.05 ** p < 0.01, compared with the model group; ## p < 0.01, compared with the GC group; && p < 0.01, compared with the GC + miR-144 simulant group.

[0028] Figure 7 This is a comparison chart of the relative expression levels of GRα, GRβ, and NF-κB mRNA in each group of the embodiments of the present invention. ** p < 0.01, compared with the model group; ## p < 0.01, compared with the GC group; && p < 0.01, compared with the GC + miR-144 simulant group. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Therapeutic effect of miR-144 inhibitor combined with glucocorticoids on ARDS rats 1. Preparation of experimental materials 1.1 Experimental animals: 6-8 week old male SD rats, weighing 180-200g, were purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd., animal license number: SCXK (Lu) 2022-0006.

[0031] 1.2 Main Reagents: Lipopolysaccharide (Shanghai Jizhi Biochemical Technology Co., Ltd.), Methylprednisolone Sodium Succinate (Shenzhen Selma Biotechnology Co., Ltd.), miR-144 mimic and miR-144 inhibitor (Guangzhou Ruibo Biotechnology Co., Ltd.), Wright-Giemsa staining solution (Shanghai Beyotime Biotechnology Co., Ltd.), Hematoxylin and Eosin (HE) staining kit (Fuzhou Feijing Biotechnology Co., Ltd.), Rat IL-6 ELISA kit (Tianjin Zancheng Technology Co., Ltd.), Rat TNF-α ELISA kit (Beijing Mindray Bio-Medical Electronics Co., Ltd.), Total RNA extraction kit (Hangzhou Borui Technology Co., Ltd.), PrimeScript RT kit (Beijing Takara Bio Inc.), 2×SYBR Green qPCR Mix (Nanjing Novizan Biotechnology Co., Ltd.), and specific primer synthesis (Sangon Biotech Co., Ltd.).

[0032] 1.3 Main instruments: electronic scale (Mettler Toledo), high-speed refrigerated centrifuge (Thermo Fisher Scientific), ultra-micro spectrophotometer (Thermo Fisher Scientific), drying oven (Hubei Huida Instrument Co., Ltd.), tissue dehydrator (Leica), tissue embedding machine (Tianjin Aihua Medical Instrument Co., Ltd.), paraffin sectioner (Leica), optical microscope (Leica), real-time fluorescence quantitative PCR instrument (ABI 7500).

[0033] 2. Model building and intervention 2.1 Preparation of lipopolysaccharide solution: Accurately weigh the lipopolysaccharide powder, dissolve it in physiological saline and make up to volume to prepare a stock solution of 10 mg / ml. Prepare and use immediately.

[0034] 2.2 Model establishment: After weighing, rats were fixed in a rat restraint device, their tails were disinfected with 75% alcohol, and 10 mg / kg lipopolysaccharide solution was injected into the tail vein to induce the ARDS model. The blank control group was injected with an equal volume of physiological saline.

[0035] 2.3 Grouping Intervention: Four hours after modeling, the model rats were randomly divided into four groups of six rats each, with six rats serving as a blank control group. The following treatments were administered to each group: - Blank control group: 1 ml of normal saline was injected intraperitoneally daily for 5 consecutive days; - Model group: Same as blank control group; - GC group: Daily intraperitoneal injection of 2 mg / kg methylprednisolone sodium succinate (dissolved in normal saline, total volume 1 ml) for 5 consecutive days; - GC+miR-144 mimic group: Daily intraperitoneal injection of 2 mg / kg methylprednisolone sodium succinate (0.5 ml) + 25 nmol / kg miR-144 mimic (0.5 ml) for 5 consecutive days; - GC+miR-144 inhibitor group: Daily intraperitoneal injection of 2 mg / kg methylprednisolone sodium succinate (0.5 ml) + 25 nmol / kg miR-144 inhibitor (0.5 ml) for 5 consecutive days; 3. Sample collection and testing Please see Figure 1 : 3.1 Weight monitoring: The weight of rats was measured daily before and after the intervention, and the data were recorded.

[0036] 3.2 Collection of Bronchoalveolar Lavage Fluid (BALF): Rats were anesthetized by intraperitoneal injection of 0.3 ml / 100 g of 10% chloral hydrate. After satisfactory anesthesia, the hair on the chest and abdomen was removed, and the skin was disinfected three times with 75% alcohol. The skin was then cut open to expose the thoracic cavity, and the tissues around the hilum were separated to expose the right hilum. The right hilum was ligated with 4-0 silk suture. An oblique incision was made in the trachea, and the catheter of the intravenous blood collection needle was inserted into the trachea and fixed with 4-0 silk suture. 5 ml of ice-cold sterile saline was injected into the trachea through the catheter. The left lung tissue was gently turned over, aspirated, and re-infused. This process was repeated three times. Finally, the lavage fluid was aspirated for alveolar lavage once. A total of three lavages were performed, and the final BALF was collected. The BALF was filtered through a single layer of gauze to remove mucus.

[0037] 3.3 Neutrophil Count: BALF samples were centrifuged at 300g for 10 minutes at 4°C. After removing the supernatant, the cell pellet was resuspended in 1ml PBS to prepare a single-cell suspension. The total nucleated cell concentration (C0.05) was calculated using a hemocytometer. totalTake a smear of the cell suspension and stain it according to the instructions for Wright-Giemsa staining solution. Calculate the percentage (N%) of neutrophils among nucleated cells under an oil immersion microscope. Absolute neutrophil count = C total ×N%.

[0038] 3.4 Detection of inflammatory factors: BALF was centrifuged at 1000g for 20 min at 4℃, the supernatant was collected and stored at -20℃ for testing, and the concentrations of IL-6 and TNF-α were detected according to the ELISA kit instructions.

[0039] 3.5 Lung wet-to-dry weight ratio determination: Rats were euthanized by cervical dislocation, and the upper lobe of the right lung was removed. The surface moisture and bloodstains were absorbed with gauze, and the wet weight was measured with an electronic scale. The lungs were dried in an oven at 80℃ for 72 hours until constant weight, and the dry weight was measured. The wet-to-dry weight ratio was calculated to assess the degree of pulmonary edema.

[0040] 3.6 Pathological Sections and Scoring: The cleaned right middle lobe of the lung was immersed in 4% formaldehyde solution and fixed for at least 24 hours, then rinsed overnight under running water. It was then subjected to a gradient dehydration process with ethanol (70%, 80%, 95% I, 95% II, anhydrous ethanol I, anhydrous ethanol II, 1 hour each), cleared with xylene (0.5 hours, twice), and embedded in paraffin (60℃ for 1 hour, twice). Sections were then prepared (4 μm thickness). The paraffin sections were then dried in a 60℃ oven for 2 hours before being immersed in... Xylene I and II, 10 min each, were used to thoroughly remove paraffin. The sections were rehydrated to distilled water consistency using a gradient of alcohols (anhydrous ethanol I, anhydrous ethanol II, 95% I, 95% II, 80%, 70%, and distilled water, 5 min each). Hematoxylin and eosin (HE) staining was performed, and the sections were mounted with neutral resin. Under an optical microscope, the following criteria were assessed: alveolar congestion or hemorrhage, neutrophil infiltration or aggregation in the alveolar cavities or vascular walls, and alveolar wall thickening or hyaline membrane formation. 0 points: mildest damage; 1 point: mild damage; 2 points: moderate damage; 3 points: severe damage; 4 points: most severe damage. The scores for each criterion were summed to obtain the final histopathological score to assess the degree of lung injury.

[0041] 3.7 RT-PCR Detection: 100 mg of right lower lung tissue was collected, and total RNA was extracted according to the instructions of the total RNA extraction kit. RNA purity and concentration were detected using a spectrophotometer. (Following PrimeScript instructions...) The RT kit instructions describe the synthesis of cDNA, amplification was performed using 2×SYBR Green qPCR Mix, and product specificity was verified by melting curve analysis. -ΔΔCt The relative expression levels of GRα, GRβ, and NF-κB were calculated using a method that normalizes the expression levels using GAPDH as an internal reference. Specific primer sequences are shown in Table 1.

[0042]

[0043] 4. Statistical Analysis All measurement data in this invention are expressed as mean ± standard deviation and analyzed using SPSS 22.0 software. The t-test was used to compare the body weight of rats before and after treatment, and also to compare the relevant data between the model group and the blank control group. One-way ANOVA was used to assess the differences in experimental results between the model group and the three different treatment groups. For post-hoc analyses, if the variances were homogeneous, the Least Significant Difference (LSD) procedure was applied; otherwise, the Games-Howell procedure was selected. P < 0.05 was considered statistically significant.

[0044] 5. Experimental Results and Analysis 5.1 Weight changes: such as Figure 2 As shown, compared with the blank control group, the rats in the model group had a significantly lower body weight and a slower recovery, indicating that inflammatory stimulation hindered the growth of rats. The GC group showed a better weight recovery than the model group, suggesting that GC had an anti-inflammatory effect. The GC + miR-144 mimic group had a slower weight recovery rate than the GC group alone. The GC + miR-144 inhibitor group had the smallest weight loss and the fastest recovery, which was significantly higher than the GC group alone, suggesting that the miR-144 inhibitor enhanced the anti-inflammatory efficacy of GC, while the miR-144 mimic had a weakening effect.

[0045] 5.2 Inflammatory suppression effect: such as Figure 3-4 As shown, compared with the blank control group, the neutrophil count, IL-6, and TNF-α concentrations in the BALF of the model group were significantly increased. Compared with the model group, the above inflammatory markers were significantly reduced in the GC group; further reduced in the GC+miR-144 inhibitor group, while inflammatory markers rebounded in the GC+miR-144 mimic group.

[0046] 5.3 Improvement in pulmonary edema: such as Figure 5 As shown, compared with the blank control group, the wet-to-dry ratio of the lungs in the model group was significantly increased, suggesting that inflammatory stimulation led to the formation of pulmonary edema. Compared with the model group, this index was significantly reduced in the GC group, indicating a reduction in pulmonary edema; the wet-to-dry ratio in the GC+miR-144 inhibitor group decreased compared with the GC group, suggesting further improvement in pulmonary edema, while the wet-to-dry ratio in the GC+miR-144 mimic group was higher than that in the GC group alone, suggesting a worsening of pulmonary edema.

[0047] 5.4 Pathological damage to lung tissue: such as Figure 6 As shown, AE is a representative HE-stained image of each group of slices (magnified 100 times), and F is a comparison of lung tissue pathological scores. Figure 6 A represents the blank control group, with intact alveolar structure and mild pathological changes; Figure 6Group B is the model group, which shows severe lung injury: alveolar hemorrhage (red arrow), neutrophil infiltration (yellow arrow), alveolar wall thickening (green arrow), and hyaline membrane formation (black arrow). These pathological changes indicate that the ARDS rat model has been successfully established, and the pathological score of this group is significantly increased. Figure 6 Group C represents the GC group, where pathological changes were less severe and scores decreased compared to the model group. Figure 6 D represents the GC+miR-144 simulant group, which shows more severe damage compared to the GC group; Figure 6 E represents the GC+miR-144 inhibitor group, which showed significantly improved lung injury compared to the GC group, suggesting that miR-144 inhibitors enhanced the sensitivity of GC treatment for ARDS.

[0048] 5.5 Molecular mechanism verification: such as Figure 7 As shown, AC represent the relative expression levels of GRα, GRβ, and NF-κB mRNA, respectively. Compared with the blank control group, GRα mRNA expression was downregulated and GRβ and NF-κB mRNA expression was upregulated in the model group. Compared with the model group, the GC group upregulated GRα and downregulated GRβ and NF-κB; the GC+miR-144 inhibitor group further downregulated GRβ and NF-κB, while the GC+miR-144 mimic group showed a rebound in GRβ and NF-κB expression, with no significant effect on GRα expression.

[0049] In summary, miR-144 inhibitors can significantly enhance the therapeutic effect of glucocorticoids on ARDS rats, and the mechanism is related to downregulating GRβ and NF-κB expression and reversing GC resistance.

Claims

1. Application of miR-144 inhibitors in the preparation of drugs that enhance the efficacy of glucocorticoid therapy for acute respiratory distress syndrome.

2. The application according to claim 1, characterized in that, The glucocorticoid is one or more of methylprednisolone sodium succinate, dexamethasone, or hydrocortisone.

3. The application according to claim 1, characterized in that, The miR-144 inhibitor includes one or more of nucleic acid inhibitors or small molecule inhibitors that specifically inhibit miR-144 expression.

4. The application according to claim 3, characterized in that, The nucleic acid inhibitor is one or more of the following: antisense oligonucleotide of miR-144, siRNA, shRNA, microRNA inhibitor, and nucleic acid aptamer.

5. The application according to claim 1, characterized in that, The drug is a combination of a miR-144 inhibitor and a glucocorticoid; the drug dosage form is one or more of the following: injection, suspension, lyophilized powder for injection, or liposome preparation.

6. The application according to claim 5, characterized in that, In the composition, the dosage of the miR-144 inhibitor is 20-30 nmol / kg, and the dosage of the glucocorticoid is 1.5-2.5 mg / kg.

7. The application according to claim 6, characterized in that, The miR-144 inhibitor is administered at a dose of 25 nmol / kg, and the glucocorticoid is methylprednisolone sodium succinate, administered at a dose of 2 mg / kg.

8. The application according to any one of claims 1-7, characterized in that, The drug exerts its sensitizing effect by downregulating GRβ and NF-κB expression, inhibiting pulmonary inflammatory response, and reducing pulmonary edema.

9. A pharmaceutical composition for enhancing the efficacy of glucocorticoid therapy for acute respiratory distress syndrome, characterized in that, The pharmaceutical composition comprises an effective amount of a miR-144 inhibitor and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition also contains glucocorticoids.

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