Treatment effect of circular RNA molecular medicine on pulmonary arterial hypertension mice

Abstract

The circular RNA molecular medicine has a treatment effect on pulmonary arterial hypertension mice. The ribonucleotide circular RNA molecule hsacirc0008828 is cloned and purified and then is modified by lipidosome, nasal administration is carried out on mice, the ribonucleotide circular RNA molecule hsacirc0008828 is effectively delivered to lung tissues of the mice, the ribonucleotide circular RNA molecule hsacirc0008828 has a treatment effect on pulmonary arterial hypertension model mice, and a novel molecular drug is provided for clinical treatment of pulmonary arterial hypertension. The circular RNA molecule disclosed by the invention is used for treating pulmonary arterial hypertension mice as a lung targeting drug.

Description

Technical field:

[0001] The invention relates to the therapeutic effect of a circular RNA molecule drug on mice with pulmonary hypertension. Background technology:

[0002] Pulmonary Artery Hypertension (PAH) is an extremely malignant and progressive disease characterized by a progressive increase in pulmonary vascular resistance and pressure due to pulmonary vasoconstriction (PAV) and pulmonary vascular remodeling (PAR). The mean pulmonary artery pressure at rest is ≥25 mmHg (mmHg), the pulmonary capillary wedge pressure (PCWP) is normal, ≤15 mmHg, and there is no chronic respiratory, cardiac or thromboembolic disease.

[0003] Pulmonary hypertension is a disease that affects the pulmonary arterioles. Its pathological feature is the progressive increase in vascular resistance caused by pulmonary vascular obstruction, which leads to increased right ventricular afterload and thus right ventricular failure. Intimal and medial hyperplasia and the resulting pulmonary vascular obstruction are considered to be key factors in the pathogenesis of pulmonary hypertension. Among them, pulmonary vasoconstriction, remodeling and thrombosis are the main causes of increased pulmonary vascular resistance in patients with pulmonary hypertension, which are related to endothelial cell dysfunction, endothelial cell-mesenchymal transition, abnormal proliferation of endothelial cells and pulmonary artery smooth muscle cells (PASMC), proportional assist ventilation (PAV) and inflammation.

[0004] At present, the treatment of pulmonary hypertension includes non-specific drugs such as oral anticoagulants and diuretics. With the study of the pathophysiological mechanism of pulmonary hypertension, drugs including prostaglandins (epoprostenol, treprostinil, iloprost), endothelin receptor antagonists (bosentan, ambrisantan) and phosphodiesterase type 5 inhibitors (sildenafil, tadalafil) have emerged one after another, which have improved its morbidity and mortality to a certain extent, but cannot fundamentally reverse pulmonary vascular remodeling (pulmonary arterial remodeling, PAR), which mainly refers to the thickening of the inner, middle and outer membranes of the muscular arteries of the small pulmonary vessels, fibrous occlusion and / or muscularization, focal hyperplasia of alveolar capillaries, etc., resulting in the 5-year survival rate of patients with pulmonary hypertension is still very low (~60%).

[0005] Among pulmonary arterial hypertension (PAH) caused by different etiologies, many are related to lung diseases and / or hypoxia (Group 3: Pulmonary hypertension due to lung diseases and / or hypoxia). The main causes are alveolar hypoxia caused by chronic lung diseases, impaired respiratory control or living at high altitudes, which are mostly related to intermittent or persistent hypoxia in the local or whole lungs. Chronic hypoxia has become a recognized cause of vascular remodeling and can aggravate pulmonary hypertension caused by other etiologies, including upper airway obstruction, pulmonary arterial hypertension (PAH) caused by pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), emphysema or interstitial lung disease. Pulmonary hypoxia response is an evolutionarily ancient and conservative stress response caused by reduced alveolar oxygen supply. Hypoxia-induced local pulmonary vasoconstriction (HPV) is an important physiological phenomenon produced by the body's stress to regulate pulmonary ventilation / perfusion, and does not cause changes in pulmonary vascular pressure. For a long time, local pulmonary vasoconstriction (HPV) has been considered an early event in the process of pulmonary hypertension, which is related to abnormal ion channel expression or function, changes in Ca2+ in pulmonary artery smooth muscle cells mediated by RhoA / Rho kinase (ROK), and reduced production of vasodilators nitric oxide (NO) and prostacyclin due to endothelial dysfunction, and increased production of vasoconstrictors (ET-1). However, long-term hypoxia can cause persistent contraction of pulmonary vessels and increase resistance. At the same time, long-term hypoxia can lead to hypoxic pulmonary vascular remodeling (hypoxic PAR, HPVR), aggravating the persistent increase in pulmonary artery pressure caused by hypoxia. Chronic hypoxia can induce intimal thickening of pulmonary arterioles, intimal fibrosis and the appearance of smooth muscle bundles, narrowing the vascular lumen and causing persistent pulmonary hypertension. Studies have shown that in addition to genetic factors, DNA damage / activation of pulmonary vascular cells, changes in non-coding RNA (non-coding RNA) expression, dysregulation of proliferation and apoptosis signaling pathways, changes in cell metabolism, metabolic flux and mitochondrial function, and endothelial cell to mesenchymal transformation are all key signaling pathways that promote pulmonary vascular remodeling (PAR). Current treatment options include drug therapy, oxygen therapy, lung transplantation, etc. Drug therapy includes vasodilators, anticoagulants, antifibrotic drugs, etc., aimed at reducing pulmonary vascular resistance and improving gas exchange. Oxygen therapy improves hypoxemia by providing high concentrations of oxygen. For patients with severe and uncontrollable conditions, lung transplantation may be the last treatment option.Currently, the therapeutic drugs on the market lack specificity for HPH, and the treatment options for HPH are limited and ineffective. Therefore, it is necessary to find targeted drug targets and therapeutic drugs based on the pathological characteristics of HPH and further study new treatment methods.

[0006] Circular RNA (circRNA) is a type of non-coding RNA molecule with a closed circular structure. It can regulate the expression of downstream target genes of microRNA (miRNA) by acting as a sponge for microRNA (miRNA). Unlike traditional linear RNA (linear RNA, containing 5' and 3' ends), circRNA molecules have a closed circular structure, are not affected by RNA exonucleases, are not easily degraded by nuclease exonucleases, and can be expressed stably and long-term in the body. Therefore, it is more stable and conservative than linear RNA. Therefore, circular RNA has unique advantages in disease diagnosis and the development of treatment strategies.

[0007] Circular RNA can be used for gene therapy of various diseases. Wang X et al. found that circular RNA delivered by exosomes has a certain inhibitory effect on the occurrence and development of colorectal cancer. Yu X et al. found that extracellular vesicle-mediated circDYM delivery alleviated CUS-induced depressive-like behavior. Wang ZY et al. found that the negative feedback of SNRK on circ-SNRK can regulate cardiac function after myocardial infarction. However, there have been no research reports on the use of circRNA as a gene therapy for the treatment of pulmonary hypertension.

[0008] In various cell studies, it was found that circRNA has a regulatory effect on cell proliferation and differentiation. The abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) leads to thickening of the vascular wall, which causes increased pulmonary vascular resistance and is the main pathological basis of pulmonary vascular remodeling. Summary of the invention:

[0009] The purpose of the present invention is to provide a circular RNA molecule, which is packaged in modified liposomes to establish a mouse lung-targeted drug delivery system and evaluate its therapeutic effect in a mouse pulmonary hypertension model.

[0010] The above purpose is achieved through the following technical solutions:

[0011] A circular RNA molecular drug has a therapeutic effect on mice with pulmonary hypertension. The ribonucleotide circular RNA molecule hsa_circ_0008828 was cloned and purified, then modified with liposomes and administered to mice nasally. It was effectively delivered to the mouse lung tissue and had a therapeutic effect on mice with pulmonary hypertension model, providing a new type of molecular drug for the clinical treatment of pulmonary hypertension.

[0012] The therapeutic effect of the circular RNA molecular drug on mice with pulmonary hypertension was evaluated by constructing the transmembrane lipid drug R8-Lip-circ0008828 based on the ribonucleotide circular RNA molecule hsa_circ_0008828 and evaluating its therapeutic effect in the mouse pulmonary hypertension model by intranasal administration.

[0013] The therapeutic effect of the circular RNA molecular drug on mice with pulmonary hypertension, the first step is to construct the liposome-modified drug R8-Lip-circ0008828, the second step is to administer R8-Lip-circ0008828 to mice intranasally, the third step is to verify the tissue distribution of the drug by in vivo imaging of mice, the fourth step is to establish a mouse pulmonary hypertension model, the fifth step is to evaluate the efficacy of R8-Lip-circ0008828 intranasally for the treatment of pulmonary hypertension in mice model: the sixth step is mouse echocardiography, the seventh step is to determine the right ventricular hypertrophy index of mice, the eighth step is hematoxylin-eosin staining of mouse organ tissues, the ninth step is fluorescent in situ hybridization of mouse lung tissues, the tenth step is immunofluorescence of mouse lung tissues, the eleventh step is immunoblot analysis of mouse lung tissues, the twelfth step is real-time fluorescence quantitative polymerase chain reaction verification of mouse lung tissues, and the thirteenth step is biological statistical analysis.

[0014] The therapeutic effect of the circular RNA molecular drug on mice with pulmonary hypertension, the first step of constructing the liposome-modified drug R8-Lip-circ0008828, step 1-cloning and purifying the hsa_circ_0008828 plasmid: constructing the expression vector of hsa_circ_0008828, adopting the conventional plasmid cloning and purification method for vector transformation and plasmid extraction, and obtaining 5 mg hsa_circ_0008828 expression plasmid;

[0015] Step 2-Preparation of modified liposomes:

[0016] ① Mix octaarginine R8 and distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide DSPE-PEG2000-Mal with chloroform and methanol at a molar ratio of 1.5:1, and then stir at room temperature for 48 hours;

[0017] ② The mixture is evaporated in a vacuum rotary evaporator to obtain a precipitate, which is then redissolved in chloroform; the supernatant distearoyl phosphatidylethanolamine-polyethylene glycol 2000-octaarginine conjugate DSPE-PEG2000-R8 is obtained by filtration, and the supernatant distearoyl phosphatidylethanolamine-polyethylene glycol 2000-octaarginine conjugate DSPE-PEG2000-R8 is evaporated under reduced pressure at 40° C. and 40 revolutions per minute to form a lipid film;

[0018] Step 3-Preparation of liposome film:

[0019] ① Mix lecithin SPC, cholesterol Chol / distearoylphosphatidylethanolamine-polyethylene glycol 2000 DSPE-PEG2000 and distearoylphosphatidylethanolamine-polyethylene glycol 2000-octaarginine conjugate DSPE-PEG2000-R8 at a molar ratio of 59:33:3:5, and add them to anhydrous ethanol;

[0020] ② The mixture was passed through a rotary evaporator to form a lipid film, and the round-bottom flask containing the lipid film was placed at 4°C for 2 hours;

[0021] Step 4 - Aqueous Dispersion:

[0022] Dissolve the lipid film with 3 mL of phosphate buffered saline (PBS) and place in a 60°C water bath for 0.75 h;

[0023] Intermittent sonication was performed using a probe sonicator at 100 watts for 0.16 h and filtered through a 0.22 μm filter membrane to ensure uniform dispersion of the liposome vesicles;

[0024] Step 5 - Packaging of hsa_circ_0008828:

[0025] 5 mg of purified hsa_circ_0008828 plasmid was mixed with blank liposomes and then incubated at 4 °C for 2 h to prepare liposome R8-Lip-circ0008828 containing 2 mg / mL hsa_circ_0008828;

[0026] Step 6 - Analysis and Characterization:

[0027] ① Use the Malvern high-sensitivity nanoparticle size analyzer to detect the particle size and polymer dispersibility index of liposome nanoparticles;

[0028] ② Use laser particle size distribution and zeta potential instrument to detect the zeta potential of nanoparticles;

[0029] ③ Use agarose gel electrophoresis to detect the encapsulation of hsa_circ_0008828;

[0030] ④ Use a transmission electron microscope to observe the morphology of the sample and take photos;

[0031] ⑤ Use mouse red blood cells to detect the biosafety of R8-Lip-circ0008828 liposome nanoparticles;

[0032] In steps 1-5, the lipid-modified R8-Lip-circ0008828 was constructed as a hsa_circ_0008828 circular RNA therapeutic drug for pulmonary arterial hypertension.

[0033] The therapeutic effect of the circular RNA molecular drug on mice with pulmonary hypertension, the second step R8-Lip-circ0008828 intranasal administration to mice, step 1, 25 mg 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindole dicarbon flower cyanine perchlorate was dissolved in 5.21 ml of anhydrous ethanol to obtain 5 millimolar 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindole dicarbon flower cyanine perchlorate DID storage solution. Phosphate buffered saline PBS was diluted 1000 times to obtain 5 micromolar 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindole dicarbon flower cyanine perchlorate DID working solution;

[0034] Step 2: The prepared R8-Lip-circ0008828 lipid-modified body and the control vector R8-Lip-PLC5 lipid-modified body were mixed with an equal volume of DID working solution and incubated at 4°C for 2 hours;

[0035] Step 3, the mice were randomly divided into 3 groups: free DID, R8-Lip-PLC5-DID, and R8-Lip-circ0008828-DID; each group of mice was anesthetized with tribromoethanol and then intranasally administered; specific administration method: the mice were anesthetized with 4% mass concentration of tribromoethanol, the mice were picked up and their bodies were placed in a supine position, and tilted at an angle of 45°, and R8-Lip-circ0008828 was slowly administered drop by drop with a sterile pipette to the outside of the mouse nostrils, 0.5 mg / mL, and the mice were inhaled with breathing, and 50 microliters were dripped into each nostril; after administration, the mice were put back into the cage and allowed to recover from anesthesia on their own;

[0036] The third step of in vivo imaging of mice to verify the tissue distribution of the drug, step 1, anesthetizing the mice 1 hour, 6 hours, 12 hours, and 24 hours after administration, placing them in a supine position in a small animal in vivo imaging instrument, selecting chemiluminescence, setting the excitation wavelength to 640 nanometers, taking pictures and recording;

[0037] Step 2: 24 hours after the injection, the mice were killed, and the main organs of the mice, including brain, heart, liver, spleen, lung, and kidney, were taken and placed in a small animal in vivo imaging device to capture in vitro fluorescence.

[0038] The therapeutic effect of the circular RNA molecule drug on mice with pulmonary hypertension, the fourth step of establishing a mouse pulmonary hypertension treatment model,

[0039] Modeling was performed using specific pathogen-free grade C57BL / 6J male mice 20-22 g according to the following steps:

[0040] Step 1, adaptive feeding: The mice were adaptively fed for 7 days to ensure their health status and body weight stability;

[0041] Step 2, grouping: mice were randomly divided into two groups;

[0042] Control group: fed in a normoxic environment, provided with adequate food and water;

[0043] Pulmonary hypertension model group: The pulmonary hypertension mouse model was established by subcutaneous injection of semasanib at a concentration of 20 mg / kg once a week, combined with hypoxia and 10% oxygen feeding for 21 days;

[0044] Step 3, anesthesia and hemodynamic and echocardiographic analysis: On the 14th and 21st days of the experiment, the mice were anesthetized with 4% mass concentration of tribromoethanol, and hemodynamic and echocardiographic analyses were performed to evaluate cardiovascular function;

[0045] Step 4, thoracotomy and organ collection: The mice were subjected to thoracotomy and perfused with phosphate buffered saline (PBS), and then their organs were collected for subsequent experiments.

[0046] The therapeutic effect of the circular RNA molecule drug on mice with pulmonary hypertension, and the fifth step of intranasal administration of R8-Lip-circ0008828 to treat the mouse model of pulmonary hypertension;

[0047] R8-Lip-circ0008828 treated pulmonary hypertension model group: The pulmonary hypertension mouse model was established according to step 4, but R8-Lip-circ0008828, 0.5 mg / mL, was intranasally administered daily from the 7th day to the 21st day according to step 3, and the mice were raised in a hypoxic environment for 21 days; the mice were intranasally administered with phosphate buffered saline (PBS) as the drug control group;

[0048] The sixth step of the mouse echocardiogram uses a small animal ultrasound instrument to record a transthoracic echocardiogram to detect the cardiac function and hemodynamics of the model mouse. The MS400 probe is used to examine small rodents to obtain echocardiographic examination images of the mouse pulmonary artery and right ventricle at a stable heart rate;

[0049] In the seventh step, the right ventricular hypertrophy index of mice is measured. The heart is dissected and the right ventricular hypertrophy is calculated by the ratio of the right ventricular weight to the right ventricular free wall weight plus the left ventricular septum weight RV / (LV+S), so as to characterize the degree of right ventricular hypertrophy.

[0050] The therapeutic effect of the circular RNA molecule drug on mice with pulmonary hypertension, the eighth step of hematoxylin-eosin staining, the mouse lung tissue is immersed in 4% paraformaldehyde for 48 hours and then dehydrated with gradient alcohol, embedded into wax blocks after being transparent in xylene, and then sliced ​​into 5-6 micron thickness, the slices of embedded mouse lung tissue are dewaxed in xylene, rehydrated with gradient alcohol, and washed with distilled water; hematoxylin staining for 0.08 hours, differentiation solution differentiation for 2-3 seconds, tap water washing to blue, eosin staining for 0.02 hours; finally, gradient alcohol dehydration, xylene transparent, neutral gum sealing, and observation and photography of pulmonary vascular reconstruction under a light microscope;

[0051] The ninth step of fluorescent in situ hybridization is to remove the paraffin-embedded lung sections of mice in xylene, and then rehydrate them with gradient ethanol and water; add proteinase K to digest the lung sections, incubate at 37°C for 0.33 hours, wash the slides with phosphate buffered saline (PBS), add denaturing solution, and denature at 78°C for 0.13 hours; add the hsa_circ_0008828 and miR-29b probe mixture to the lung tissue, incubate at 37°C overnight; discard the hybridization solution, wash three times with phosphate buffered saline (PBS), and then drop KCNB1 rabbit antibody on the tissue, incubate at 4°C in the dark overnight. Add horseradish peroxidase-labeled anti-rabbit antibody and incubate for 1 hour, wash with phosphate buffered saline (PBS), and then drop 4',6-diamidino-2-phenylindole and incubate for 0.17 hours; add anti-fluorescence quenching sealing agent, cover with a cover glass, seal with nail polish, and detect with a fluorescence microscope.

[0052] The therapeutic effect of the circular RNA molecule drug on mice with pulmonary hypertension, the tenth step of immunofluorescence, the mouse paraffin-embedded lung sections are dewaxed and rehydrated, the slides are boiled at 98°C for 0.17 hours with 0.01 mol ethylenediaminetetraacetic acid antigen repair solution for antigen repair; α-smooth muscle actin primary antibody is added to the tissue, and incubated at 4°C overnight in the dark; horseradish peroxidase HRP-labeled secondary antibody is added for incubation for 1 hour, DPAI is added for incubation in the dark for 0.17 hours, after PBS washing, anti-fluorescence quenching sealing agent is added, coverslips are covered, nail polish is used to seal the slides, and fluorescence microscopy is used for detection;

[0053] In the eleventh step of immunoblotting analysis, mouse lung tissue was lysed on ice using 1 mL of protein lysis buffer containing 1% phenylmethylsulfonyl fluoride, total protein was extracted, quantified using a BCA protein concentration assay kit, and then the protein was separated by electrophoresis in an SDS-PAGE gel, and the protein was transferred to a polyvinylidene fluoride membrane using a transfer apparatus, and the PVDF membrane was blocked with a 5% mass concentration skim milk blocking solution at room temperature for 2 hours, a primary antibody was added, and the membrane was incubated overnight at 4°C. After washing the membrane, an HRP-labeled goat anti-rabbit immunoglobulin G secondary antibody was added and incubated at room temperature for 1 hour; an ECL enhanced chemiluminescence colorimetric reagent was added dropwise to the PVDF membrane, and finally a gel imager was used for detection and analysis and photographing and recording.

[0054] The therapeutic effect of the circular RNA molecular drug on mice with pulmonary hypertension, the twelfth step of the real-time fluorescent quantitative polymerase chain reaction, extracting total RNA from mouse lung tissue, using a complementary DNA reverse transcription kit to reverse RNA into cDNA, designing primers to amplify hsa_circ_0008828 and KCNB1, respectively, and amplifying β-actin as a control;

[0055] In the thirteenth step of statistical analysis, data analysis was performed using unpaired t-test for two-group comparison and one-way analysis of variance, and the minimum P value of 0.05 was selected as the significance level.

[0056] Beneficial effects:

[0057] 1. The modified R8-Lip-circ0008828 particles of the present invention have good dispersion stability, they repel each other and are not easy to aggregate.

[0058] 2. The present invention obtained an R8-Lip-circ0008828 sample with an encapsulation efficiency of 86.3% and ensured the high quality of the liposomes.

[0059] 3. The modified R8-Lip-circ0008828 of the present invention is targeted and dispersed in the lungs of mice after nasal administration.

[0060] 4. The modified R8-Lip-circ0008828 of the present invention has a significant therapeutic effect on mice with pulmonary arterial hypertension. Description of the drawings:

[0061] Attached Figure 1 This is the physical and chemical property analysis diagram A of R8-Lip-circ0008828 of the present invention.

[0062] Attached Figure 2 This is the physical and chemical property analysis diagram B of R8-Lip-circ0008828 of the present invention.

[0063] Attached Figure 3This is the physical and chemical property analysis diagram C of R8-Lip-circ0008828 of the present invention.

[0064] Attached Figure 4 This is the physical and chemical property analysis diagram D of R8-Lip-circ0008828 of the present invention.

[0065] Attached Figure 5 FIGE is the encapsulation efficiency experiment of the present invention, which characterizes the encapsulation of R8-Lip-circ0008828 liposome nanoparticles.

[0066] Attached Figure 6 FIG. F is the encapsulation efficiency experiment of the present invention characterizing the encapsulation of R8-Lip-circ0008828 liposome nanoparticles.

[0067] Attached Figure 7 Figure A is a transmission electron microscopy image of the R8-Lip-circ0008828 liposome of the present invention.

[0068] Attached Figure 8 Figure B is the 7-day stability diagram of R8-Lip-circ0008828 of the present invention in PBS.

[0069] Attached Fig. 9 Figure A is an image and quantitative analysis of the hemolytic experiment of R8-Lip-circ0008828 at different concentrations of the present invention.

[0070] Attached Fig.10 It is a graph of the mouse erythrocyte hemolysis test of the present invention.

[0071] Attached Fig.11 Figure A is an in vitro fluorescence image of the lung tissue of a control mouse that was intranasally administered with Free-DID, the lung tissue of a mouse that was intranasally administered with R8-Lip-PLC5 liposomes, and the lung tissue of a mouse that was intranasally administered with R8-Lip-circ0008828 liposomes.

[0072] Attached Fig.12 Figure B is the in vitro fluorescent image of the isolated brain, heart, liver, spleen, lung and kidney of each group of mice in the present invention.

[0073] Attached Fig.13 Figure C is the in vivo imaging of each group of mice of the present invention at 1h, 6h, 12h and 24h.

[0074] Attached Fig.14 These are representative fluorescent images of α-SMA, hsa_circ_0008828, and DAPI in pulmonary artery sections of each group of mice in the present invention.

[0075] Attached Fig.15Figure A is the in vitro fluorescent image of the isolated brain, heart, liver, spleen, lung and kidney of each group of mice in the present invention.

[0076] Attached Fig.16 FIG. A is a statistical result of the ratio of the right ventricle weight to the left ventricle plus the ventricular septum weight (RV / (LV+S)) of the present invention.

[0077] Attached Fig.17 Figure B is a representative image of HE staining of the present invention.

[0078] Attached Fig.18 Figure C is the statistical result of HE staining of the present invention.

[0079] Attached Fig.19 Figure D is a representative image of α-SMA immunofluorescence (green) of the present invention.

[0080] Attached Fig. 20 FIG. E is the statistical result of α-SMA immunofluorescence (green) of the present invention.

[0081] Attached Fig.21 It is a representative image and statistical graph A of the present invention for detecting pulmonary artery hemodynamics and right ventricular function of pulmonary hypertension mice by small animal transthoracic echocardiography.

[0082] Attached Fig. 22 It is the pulmonary artery blood flow acceleration time (PAT) graph B of the present invention.

[0083] Attached Fig.23 Graph C is the ratio of pulmonary artery acceleration time to pulmonary artery ejection time (PAT / PET) of the present invention.

[0084] Attached Fig.24 FIG. D is the maximum pulmonary artery blood flow velocity (PVmax) of the present invention.

[0085] Attached Fig.25 It is the pulmonary artery velocity time integral (PAVIT) graph E of the present invention.

[0086] Attached Fig.26 FIG. F is the tricuspid annular systolic displacement (TAPSE) of the present invention.

[0087] Attached Fig. 27 Graph G of right ventricular output (CO) of the present invention.

[0088] Attached Fig.28 Figure A is the expression level of hsa_circ_0008828 detected by qRT-PCR of the present invention.

[0089] Attached Fig.29 FIG. B is the expression level of miR-29b detected by qPCR of the present invention.

[0090] Attached Fig.30 FIG. C is the expression level of KCNB1 detected by qPCR of the present invention.

[0091] Attached Fig.31 Figure D is a representative confocal image of hsa_circ_0008828, miR-29b, KCNB1 and DAPI in the pulmonary artery sections of each group of mice in the present invention.

[0092] Attached Fig.32 FIG. 4 is the fluorescence quantitative graph E of the present invention. Fig.33 This is the fluorescence quantitative graph F of the present invention.

[0093] Attached Fig.34 This is the fluorescence quantitative graph G of the present invention. Specific implementation method:

[0094] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0095] Embodiment 1:

[0096] A circular RNA molecular drug has a therapeutic effect on mice with pulmonary hypertension. The circular RNA (ribonucleotide) molecule hsa_circ_0008828 was cloned and purified, modified with liposomes, and administered to mice nasally. It was effectively delivered to the mouse lung tissue and had a therapeutic effect on pulmonary hypertension model mice, providing a new type of molecular drug for the treatment of clinical pulmonary hypertension. After circNFXL1 was modified with locked nucleic acid, it was administered to mice nasally to effectively target the mouse lung tissue.

[0097] Embodiment 2:

[0098] The therapeutic effect of the circular RNA molecular drug described in Example 1 on mice with pulmonary hypertension. Based on the circular RNA molecule hsa_circ_0008828, a transmembrane lipid drug R8-Lip-circ0008828 (R8, abbreviation of octaarginine; Lip, abbreviation of liposome) was constructed, and its therapeutic effect in the mouse pulmonary hypertension model was evaluated by intranasal administration to mice.

[0099] Embodiment 3:

[0100] The therapeutic effect of the circular RNA molecular drug described in Example 1 or 2 on mice with pulmonary hypertension, the first step is to construct the liposome-modified drug R8-Lip-circ0008828, the second step is to administer R8-Lip-circ0008828 to mice intranasally, the third step is to verify the tissue distribution of the drug by in vivo imaging of mice, the fourth step is to establish a mouse pulmonary hypertension model, the fifth step is to evaluate the efficacy of R8-Lip-circ0008828 intranasally for the treatment of pulmonary hypertension in mice (the evaluation index is as follows: the sixth step is mouse echocardiography, the seventh step is mouse right ventricular hypertrophy index determination, the eighth step is mouse organ tissue hematoxylin-eosin staining (HE staining for short), the ninth step is mouse lung tissue fluorescence in situ hybridization (Fluorescence in situ Hybridization, FISH), the tenth step is mouse lung tissue immunofluorescence (Immunofluorescence, IF), the eleventh step is mouse lung tissue immunoblot analysis (western blot), the twelfth step is mouse lung tissue real-time quantitative polymerase chain reaction (real-time quantitative Polymerase Chain Reaction, qRT-PCR) verification, step thirteen biological statistical analysis.

[0101] Embodiment 4:

[0102] The therapeutic effect of the circular RNA molecule drug described in Example 3 on mice with pulmonary hypertension, the first step of constructing the liposome-modified drug R8-Lip-circ0008828, step 1-cloning and purification of hsa_circ_0008828 plasmid: based on the previous research of the authors, the expression vector of hsa_circ_0008828 was constructed, and the conventional plasmid cloning and purification method was adopted for vector transformation and plasmid extraction to obtain 5 mg of hsa_circ_0008828 expression plasmid;

[0103] Step 2-Preparation of modified liposomes:

[0104] ① Mix octaarginine R8 and distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide DSPE-PEG2000-Mal with chloroform and methanol at a molar ratio of 1.5:1, and then stir at room temperature for 48 hours;

[0105] ② The mixture is evaporated in a vacuum rotary evaporator to obtain a precipitate, which is then redissolved in chloroform; the supernatant distearoyl phosphatidylethanolamine-polyethylene glycol 2000-octaarginine conjugate DSPE-PEG2000-R8 is obtained by filtration, and the supernatant distearoyl phosphatidylethanolamine-polyethylene glycol 2000-octaarginine conjugate DSPE-PEG2000-R8 is evaporated under reduced pressure at 40° C. and 40 revolutions per minute to form a lipid film;

[0106] Step 3-Preparation of liposome film:

[0107] ① Mix lecithin SPC, cholesterol Chol / distearoylphosphatidylethanolamine-polyethylene glycol 2000 DSPE-PEG2000 and distearoylphosphatidylethanolamine-polyethylene glycol 2000-octaarginine conjugate DSPE-PEG2000-R8 at a molar ratio of 59:33:3:5, and add them to anhydrous ethanol;

[0108] ② The mixture was passed through a rotary evaporator to form a lipid film, and the round-bottom flask containing the lipid film was placed at 4°C for 2 hours;

[0109] Step 4 - Aqueous Dispersion:

[0110] Dissolve the lipid film with 3 mL of phosphate buffered saline (PBS) (pH = 7.4) and incubate in a 60 °C water bath for 0.75 h;

[0111] Intermittent sonication was performed using a probe sonicator at 100 watts (W) for 0.16 h and filtered through a 0.22 micron (μm) filter membrane to ensure uniform dispersion of the liposome vesicles;

[0112] Step 5 - Packaging of hsa_circ_0008828:

[0113] 5 mg of purified hsa_circ_0008828 plasmid was mixed with blank liposomes and then incubated at 4 °C for 2 h to prepare liposome R8-Lip-circ0008828 containing 2 mg / mL hsa_circ_0008828;

[0114] Step 6 - Analysis and Characterization:

[0115] ① Use the Malvern high-sensitivity nanoparticle size analyzer (model ZEN1690) to detect the particle diameter (Particle Diameter, PD) and polymer dispersity index (Polydispersity Index, PDI) of liposome nanoparticles;

[0116] ② Use laser particle size distribution and zeta potential instrument to detect the zeta potential of nanoparticles;

[0117] ③ The encapsulation of hsa_circ_0008828 was detected using agarose gel electrophoresis (SDS-PAGE);

[0118] ④ Use a transmission electron microscope, model HT-7800, to observe the morphology of the samples and take photos;

[0119] ⑤ Use mouse red blood cells to detect the biosafety of R8-Lip-circ0008828 liposome nanoparticles;

[0120] In steps 1-5, the lipid-modified R8-Lip-circ0008828 was constructed as a hsa_circ_0008828 circular RNA therapeutic drug for pulmonary arterial hypertension.

[0121] Embodiment 5:

[0122] The therapeutic effect of the circular RNA molecular drug described in Example 3 on mice with pulmonary arterial hypertension, the second step R8-Lip-circ0008828 intranasally administered to mice, step 1, 25 mg 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindodicarbocyanine perchlorate (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine-4-Chlorobenzenesu lfonate Salt, DID) was dissolved in 5.21 ml of anhydrous ethanol to obtain 5 millimolar (mmol / L, mM) 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindodicarbocyanine perchlorate DID storage solution. Phosphate buffered saline (PBS) was diluted 1000 times to obtain a 5 μmol / L (μM) 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindolyl dicarbonyl cyanine perchlorate (DID) working solution;

[0123] Step 2: The prepared R8-Lip-circ0008828 lipid-modified body and the control vector R8-Lip-PLC5 lipid-modified body were mixed with an equal volume of DID working solution and incubated at 4°C for 2 hours;

[0124] Step 3, the mice were randomly divided into 3 groups: free DID, R8-Lip-PLC5-DID, and R8-Lip-circ0008828-DID. Each group of mice was anesthetized with tribromoethanol and then intranasally administered; specific administration method: the mice were anesthetized with 4% mass concentration (w / v) tribromoethanol, the mice were picked up and their bodies were lying on their backs, and tilted at a 45° angle, and R8-Lip-circ0008828 was slowly administered drop by drop with a sterile pipette to the outside of the mouse nostrils, 0.5 mg / mL, so that it could be inhaled with breathing, and 50 microliters (μL) were dripped into each nostril; after administration, the mice were put back in the cage and allowed to recover from anesthesia on their own;

[0125] The third step of in vivo imaging of mice to verify the tissue distribution of the drug, step 1, anesthetizing the mice 1 hour, 6 hours, 12 hours, and 24 hours after administration, placing them in a supine position in a small animal in vivo imaging instrument, selecting chemiluminescence, setting the excitation wavelength to 640 nanometers, taking pictures and recording;

[0126] Step 2: 24 hours after the injection, the mice were killed, and the main organs of the mice, including brain, heart, liver, spleen, lung, and kidney, were taken and placed in a small animal in vivo imaging device to capture in vitro fluorescence.

[0127] This experiment aims to simulate the pathological conditions of pulmonary hypertension and test whether R8-Lip-circ0008828 has therapeutic potential; the main focuses of the study include cardiovascular function, the establishment of a pulmonary hypertension model, and the therapeutic effect of R8-Lip-circ0008828; these results help to gain a deeper understanding of the effectiveness and mechanism of this treatment method.

[0128] Embodiment 6:

[0129] The therapeutic effect of the circular RNA molecule drug described in Example 3 on mice with pulmonary hypertension, the fourth step of establishing a mouse pulmonary hypertension treatment model,

[0130] Modeling was performed using specific pathogen-free grade C57BL / 6J male mice 20-22 g according to the following steps:

[0131] Step 1, adaptive feeding: The mice were adaptively fed for 1 week to ensure their health status and body weight stability;

[0132] Step 2, grouping: mice were randomly divided into two groups;

[0133] Control group: fed in a normoxic environment, provided with adequate food and water;

[0134] Pulmonary hypertension model group: The pulmonary hypertension mouse model was established by subcutaneous injection of Sugen5416 (SU5416) at a concentration of 20 mg / kg once a week, combined with hypoxia and 10% oxygen feeding for 21 days;

[0135] Step 3, anesthesia and hemodynamic and echocardiographic analysis: On the 14th and 21st days of the experiment, the mice were anesthetized with 4% mass concentration (w / v) tribromoethanol, and hemodynamic and echocardiographic analyses were performed to evaluate cardiovascular function;

[0136] Step 4, thoracotomy and organ collection: The mice were subjected to thoracotomy and perfused with phosphate buffered saline (PBS), and then their organs were collected for subsequent experiments.

[0137] Embodiment 7:

[0138] The therapeutic effect of the circular RNA molecule drug described in Example 3 on mice with pulmonary hypertension, and the fifth step of intranasal administration of R8-Lip-circ0008828 to treat the mouse model of pulmonary hypertension;

[0139] R8-Lip-circ0008828 treated pulmonary hypertension model group: The pulmonary hypertension mouse model was established according to step 4, but R8-Lip-circ0008828, 0.5 mg / mL, was intranasally administered daily from the 7th day to the 21st day according to step 3, and the mice were raised in a hypoxic environment for 21 days; the mice were intranasally administered with phosphate buffered saline (PBS) as the drug control group;

[0140] The sixth step of the mouse echocardiogram uses a small animal ultrasound instrument to record a transthoracic echocardiogram to detect the cardiac function and hemodynamics of the model mouse. The model MS400 probe is used to examine small rodents, and the echocardiogram examination images of the mouse pulmonary artery and right ventricle are obtained at a stable heart rate;

[0141] In the seventh step, the right ventricular hypertrophy index of mice is measured. The heart is dissected and the right ventricular hypertrophy is calculated by the ratio of the right ventricular weight to the right ventricular free wall weight plus the left ventricular septum weight RV / (LV+S), so as to characterize the degree of right ventricular hypertrophy.

[0142] Embodiment 8:

[0143] The therapeutic effect of the circular RNA molecule drug described in Example 3 on mice with pulmonary hypertension, the eighth step of hematoxylin-eosin staining (HE staining), the mouse lung tissue was immersed in 4% paraformaldehyde (4% PFA) and fixed for 48 hours and then dehydrated with gradient alcohol, embedded into wax blocks after being transparent in xylene, and then sliced ​​with a thickness of 5-6 microns (μm), the slices of embedded mouse lung tissue were dewaxed in xylene, rehydrated with gradient alcohol, and washed with distilled water; hematoxylin staining for 0.08 hours, differentiated with differentiation solution for 2-3 seconds, rinsed with tap water to return to blue, and eosin staining for 0.02 hours; finally, gradient alcohol dehydration was used, xylene was transparent, and neutral gum was used to seal the slices, and the reconstruction of pulmonary blood vessels was observed and photographed under a light microscope;

[0144] The ninth step of fluorescence in situ hybridization (FISH) is to remove the paraffin-embedded lung sections of mice in xylene, and then rehydrate them with gradient ethanol and water; add proteinase K to digest the lung sections, incubate at 37°C for 0.33 hours, wash the slides with phosphate buffered saline (PBS), add denaturing solution, and denature at 78°C for 0.13 hours; add the hsa_circ_0008828 and miR-29b probe mixture to the lung tissue and incubate at 37°C overnight; discard the hybridization solution, wash three times with phosphate buffered saline (PBS), and then add KCNB1 rabbit antibody (referred to as "primary antibody") to the tissue and incubate overnight at 4°C in the dark. Horseradish peroxidase (HRP)-labeled anti-rabbit antibody (referred to as "secondary antibody") was added and incubated for 1 hour. After washing with phosphate buffered saline (PBS), 4',6-diamidino-2-phenylindole (DAPI) was added and incubated for 0.17 hours. Anti-fluorescence quenching mounting medium was added, coverslips were covered, nail polish was used to seal the slides, and fluorescence microscopy was used for examination.

[0145] Embodiment 9:

[0146] The therapeutic effect of the circular RNA molecule drug described in Example 3 on mice with pulmonary hypertension, the tenth step of immunofluorescence, the paraffin-embedded lung sections of mice were dewaxed and rehydrated, the slides were boiled at 98°C for 0.17 hours with 0.01 molar ethylenediaminetetraacetic acid (1×EDTA) antigen repair solution for antigen repair; α-smooth muscle actin (α-SMA) primary antibody was added to the tissue and incubated overnight at 4°C in the dark; horseradish peroxidase HRP-labeled secondary antibody was added and incubated for 1 hour, DPAI was added and incubated in the dark for 0.17 hours, after PBS washing, anti-fluorescence quenching sealing agent was added, coverslips were covered, nail polish was used to seal the slides, and fluorescence microscopy was used for detection;

[0147] In the eleventh step of immunoblotting analysis (western blot), mouse lung tissue was lysed on ice using 1 mL of protein lysis buffer (Radio Immunoprecipitation Assay Lysis Buffer, RIPA lysis buffer) containing 1% phenylmethylsulfonyl fluoride (PMSF), total protein was extracted, quantified using a BCA protein concentration determination kit, and then the protein was separated by electrophoresis in an SDS-PAGE gel, and the protein was transferred to a polyvinylidene fluoride membrane (PVDF membrane) using a transfer membrane instrument. The PVDF membrane was blocked with a 5% mass concentration skim milk blocking solution at room temperature for 2 hours, a primary antibody was added, and the membrane was incubated overnight at 4°C. After washing the membrane, HRP-labeled goat anti-rabbit immunoglobulin G (immunoglobulin G, IgG) secondary antibody was added and incubated at room temperature for 1 hour; ECL enhanced chemiluminescence (Enhanced Chemiluminescence, ECL) colorimetric reagent was added dropwise on the PVDF membrane, and finally a gel imager was used for detection and analysis and photographing and recording.

[0148] Embodiment 10:

[0149] The therapeutic effect of the circular RNA molecular drug described in Example 3 on mice with pulmonary hypertension is characterized by: the twelfth step is real-time quantitative polymerase chain reaction (qRT-PCR), extracting total RNA from mouse lung tissue, using a complementary DNA (cDNA) reverse transcription kit to reverse the RNA into cDNA, and designing primers to amplify hsa_circ_0008828 and KCNB1, respectively, to amplify β-actin as a control;

[0150] In the thirteenth step of statistical analysis, data analysis was performed using unpaired t-test for two-group comparison and one-way analysis of variance, and the minimum P value (Probability Value, P value) of 0.05 was selected as the significance level.

[0151] Embodiment 11:

[0152] The therapeutic effect of the targeted molecular drugs described in the above examples on mice with pulmonary hypertension,

[0153] The inventors' previous work discovered a unique circRNA expression pattern in PAH patients and screened out hsa_circ0008828 as a candidate biomarker for PAH patients, and its dysregulation can promote abnormal proliferation of pulmonary artery smooth muscle cells.

[0154] By comparing the circRNA expression profiles of PH patients with those of normal physical examination subjects, we found circRNA dysregulation in 158 PH patients using microarray technology. Quantitative analysis further confirmed that in a large number of subjects, hsa_circ_0008828 was significantly downregulated in PH patients, with the largest amplitude, and had extremely high diagnostic value. Further mechanism studies showed that hsa_circ_0008828 is a key regulatory factor in hypoxia-related PAH. Hypoxia can cause downregulation of hsa_circ_0008828 expression, thereby inhibiting K+ channel activity, inducing abnormal proliferation, apoptosis, and migration of hPASMCs, and promoting pulmonary vascular remodeling, suggesting that hsa_circ_0008828 has a protective effect in hypoxic pulmonary vascular remodeling (HPVR).

[0155] In recent years, preclinical and clinical data have suggested the potential of RNA therapy for treating diseases. This application uses circRNA therapy to construct a drug delivery system (R8-lip-circ0008828) with hsa_circ_0008828 as the molecular drug to study its effect and improvement on the course of pulmonary arterial hypertension, which can provide new ideas for the treatment and diagnosis of clinical pulmonary arterial hypertension.

[0156] Embodiment 12:

[0157] The therapeutic effect of the targeted molecular drugs described in the above examples on mice with pulmonary hypertension,

[0158] Experimental Results

[0159] (1) Characterization of R8-Lip-circ0008828 particle size and zeta potential

[0160] After dilution with PBS, we used the Malvern high-sensitivity nanoparticle size analyzer ZEN1690 to measure the particle diameter (PD) and polymer dispersity index (PDI) of R8-Lip-circ0008828 nanoparticles. At the same time, we used a laser particle size distribution analyzer and a zeta potential analyzer to measure the zeta potential of the nanoparticles.

[0161] The results showed that the average particle size of R8-Lip-circ0008828 was 154.2±4.3 nanometers, which is consistent with the size standard of drug delivery particles (studies have shown that when the average particle size of liposome nanoparticles is less than 200 nanometers, they can be used for injection). In addition, the PDI was 0.2±0.01, indicating that it has good dispersibility. In addition, the absolute value of the zeta potential exceeded 20, which indicates that the modified R8-Lip-circ0008828 particles have good dispersion stability, they repel each other and are not easy to aggregate. (As shown in the attached Figure 1 , Attachment Figure 2 , Attachment Figure 3 , Attachment Figure 4 )

[0162] Attached Figure 1 .Physical and chemical properties analysis of R8-Lip-circ0008828 A, Particle size of blank liposome R8-Lip and liposome R8-Lip-circ0008828 loaded with circNFLX1. Figure 2 .R8-Lip-circ0008828Physical and chemical properties analysis B, particle size distribution range of B plasmid. Figure 3 .Physical and chemical properties analysis of R8-Lip-circ0008828C, CPDI index of blank liposomes R8-Lip and liposomes loaded with circNFLX1. Figure 4 .Physical and chemical properties analysis of R8-Lip-circ0008828D, Dzeta potential of blank liposomes R8-Lip and liposomes R8-Lip-circ0008828 encapsulating circNFLX1.

[0163] (2) R8-Lip-circ0008828 loading capacity

[0164] According to the Chinese Pharmacopoeia, the encapsulation efficiency of liposomes used for drug delivery should be no less than 80%. After agarose gel electrophoresis screening, we obtained the R8-Lip-circ0008828 sample with an encapsulation efficiency of 86.3% (see attached Figure 5 , Attachment Figure 6 ). Therefore, we confirm that R8-Lip-circ0008828 can be used for drug delivery and recommend using it at a concentration between 0.5 mg / mL and 1 mg / mL. This meets the requirements of the Chinese Pharmacopoeia and ensures the high quality and effectiveness of the liposomes.

[0165] Attached Figure 5 E. Encapsulation efficiency experiment characterized the encapsulation of R8-Lip-circ0008828 liposome nanoparticles. Figure 6, F. The encapsulation efficiency experiment characterized the encapsulation of R8-Lip-circ0008828 liposome nanoparticles (Figure F).

[0166] (3) Morphological characteristics of R8-Lip-circ0008828

[0167] In order to characterize the morphological characteristics of R8-Lip-circ0008828, we used transmission electron microscopy to observe the R8-Lip-circ0008828 liposomes. The observation results showed that the R8-Lip-circ0008828 liposome nanoparticles showed a round morphology and they were well dispersed. This finding further demonstrated the high quality and applicability of the R8-Lip-circ0008828 liposomes for various drug delivery applications. (See Appendix Figure 7 Transmission electron microscopy image of R8-Lip-circ0008828 liposomes)

[0168] (4) Seven-day stability of R8-Lip-circ0008828

[0169] During the storage of liposome preparations, liposomes are prone to aggregation and fusion, which may cause changes in the average particle size and particle size distribution of liposomes. Therefore, maintaining the stability of liposomes is crucial for their practical application as drug carriers. To evaluate the stability of R8-Lip-circ0008828, we stored it at 4°C and measured and recorded its average particle size, zeta potential, and PDI every day for 7 consecutive days to characterize its stability.

[0170] The experimental results showed that R8-Lip-circ0008828 could maintain stability in phosphate buffered saline (PBS) for 7 consecutive days (see attached Figure 8 7-day stability of R8-Lip-circ0008828 in PBS). This finding verifies that R8-Lip-circ0008828 liposomes have good stability and provides a solid foundation for its application in the field of drug delivery.

[0171] (5) Safety of R8-Lip-circ0008828

[0172] To evaluate the safety of R8-Lip-circ0008828 liposomes, we performed a hemolysis test and added them to a 2% red blood cell suspension of mice to detect potential hemolysis. The test results showed that when the plasmid concentration was 1 mg / ml, the hemolysis of R8-Lip-circ0008828 was 5.18%. When the plasmid concentration was 0.5 mg / ml, the hemolysis of R8-Lip-circ0008828 was 3.47% (see attached Fig. 9 , Attachment Fig.10 ). Therefore, it can be concluded that R8-Lip-circ0008828 liposomes exhibited good biosafety when the concentration was less than 1 mg / mL. This finding indicates that R8-Lip-circ0008828 liposomes do not induce significant hemolytic reactions at appropriate concentrations, which provides important support for its safety in biomedical applications.

[0173] Fig. 9 Images and quantitative analysis of hemolysis experiments with different concentrations of R8-Lip-circ0008828. Fig.10 The mouse red blood cell hemolysis test is used to evaluate the biocompatibility and safety of the preparation. When the hemolysis rate is less than 5%, the preparation is considered to be biosafe.

[0174] (6) R8-Lip-circ0008828 was effectively delivered to mouse lung tissue.

[0175] According to the results of small animal in vivo imaging, compared with the Free-DID group and the R8-Lip-PLC5 group, both tail vein and intranasal administration of R8-Lip-circ0008828-DID showed strong signals in the mouse lung tissue, indicating that R8-Lip-circ0008828 liposomes have good targeting to the lungs. The fluorescence of intranasal administration is stronger than that of tail vein administration, indicating that intranasal administration of R8-Lip-circ0008828 liposomes has better lung targeting.

[0176] Fig.11 , R8 liposomes were injected into the tail vein of mice. 12 hours later, the lung tissue imaging results showed that the R8-modified liposomes had a strong fluorescence intensity and had a strong lung targeting. A. In vitro fluorescence images of the lung tissue of the control mouse injected with Free-DID by the tail vein, the lung tissue of the mouse injected with R8-Lip-PLC5 liposomes by the tail vein, and the lung tissue of the mouse injected with R8-Lip-circ0008828 liposomes by the tail vein. Fig.12 A. R8 liposomes were injected into the tail vein of mice. 12 hours later, the lung tissue imaging results showed that the R8-modified liposomes had a strong fluorescence intensity and a strong lung targeting ability. B. In vitro fluorescence images of the brain, heart, liver, spleen, lung, and kidney of mice in each group. Fig.13 C. R8 liposomes were injected into the tail vein of mice. 12 hours later, the lung tissue imaging results showed that the R8-modified liposomes had a strong fluorescence intensity and a strong lung targeting ability. C. In vivo imaging of mice in each group at 1h, 6h, 12h and 24h.

[0177] Mice were intranasally injected with R8-modified liposome nanoparticles. 12 hours later, lung tissue imaging results showed that intranasal administration of R8-modified liposomes had stronger fluorescence intensity than tail vein administration and better lung targeting. Fig.14 , In vitro fluorescence images of lung tissues of control mice administered intranasally with Free-DID, lung tissues of mice administered intranasally with R8-Lip-PLC5 liposomes, and lung tissues of mice administered intranasally with R8-Lip-circ0008828 liposomes. Fig.15 , A. In vitro fluorescence images of the brain, heart, liver, spleen, lung, and kidney of mice in each group. Fig.16 , the statistical results of the ratio of right ventricular mass to left ventricular plus septal mass (RV / (LV+S)) are shown in Figure A.

[0178] (7) R8-Lip-circ0008828 is effectively delivered to the pulmonary vasculature

[0179] Through immunofluorescence experiments, we observed that the expression position of circ0008828 was consistent with that of α-smooth muscle actin, and after the administration of R8-Lip-circ0008828, the expression of circ0008828 in the pulmonary vessels of mice in the circ0008828 treatment group increased significantly. This indicates that the administration of R8-Lip-circ0008828 can effectively deliver circ0008828 to the pulmonary vessels of mice, confirming the effectiveness of R8-Lip-circ0008828 as a delivery vector, especially the ability to target circ0008828 to the pulmonary vessels. This is of great significance for the treatment and research of pulmonary vascular-related diseases.

[0180] Colocalization of α-SMA and circ0008828, and the expression level of circ0008828 in mouse pulmonary vasculature. Fig.17 , B. Representative fluorescent images of α-SMA (green), circ0008828 (red), and DAPI (blue) in pulmonary artery sections of each group of mice. Fig.18 Statistical results of staining are shown in Figure C. (8) R8-lip-circ0008828 effectively inhibited right ventricular hypertrophy in mice with pulmonary hypertension

[0181] Further, we used the right ventricular hypertrophy index (i.e., the ratio of the right ventricle to the left ventricle plus the septum) to evaluate the cardiac status of mice. The experimental results showed that the RV / LV+S ratio of mice in the SuHx pathological group was significantly increased, indicating right ventricular hypertrophy. However, the administration of R8-Lip-circ0008828 significantly reduced this ratio, indicating that it effectively inhibited right ventricular hypertrophy in mice with pulmonary hypertension. This result suggests that R8-Lip-circ0008828 has a potential therapeutic effect on the treatment of right ventricular hypertrophy associated with pulmonary hypertension, providing an option for the treatment of related cardiovascular diseases.

[0182] Fig.19 , the ratio of right ventricular weight to left ventricular plus septal weight (RV / (LV+S)), characterizing right ventricular hypertrophy. A. Mice were randomly divided into three groups. (i) control group (nor); (ii) SuHx pathological group (exposed to chronic hypoxia [10% oxygen] for 3 weeks, subcutaneous injection of Sugen5416 [20 mg / kg); (iii) SuHx+R8-Lip-circ0008828 administration group (circ).

[0183] (9) R8-lip-circ0008828 effectively inhibited the pulmonary vascular remodeling process in mice with pulmonary hypertension

[0184] The results of HE staining showed that the pulmonary arterioles of mice in the pulmonary hypertension model group showed obvious stenosis and remodeling. However, after the administration of R8-Lip-circ0008828, the degree of remodeling of the pulmonary arterioles was significantly reduced. In addition, the results of α-SMA immunofluorescence showed that the degree of muscularization of the pulmonary arterioles in the mice in the pulmonary hypertension model group was significantly increased, while the degree of muscularization of the pulmonary arterioles in the R8-Lip-circ0008828 administration group was reduced. Therefore, the administration of R8-Lip-circ0008828 successfully inhibited the pulmonary vascular remodeling and muscularization process in mice with pulmonary hypertension. This finding highlights the potential therapeutic effect of R8-Lip-circ0008828, especially in dealing with pulmonary vascular structural problems related to pulmonary hypertension, which has important clinical significance.

[0185] Fig. 20 Representative images of HE staining and α-SMA immunofluorescence (green). A. Representative HE staining of pulmonary arterioles, B. Representative immunofluorescence staining images of α-smooth muscle actin (α-SMA); C. Vascular wall thickness / total pulmonary arteriole thickness (percentage thickness); D. Percentage of wall thickness under immunofluorescence.

[0186] (10) R8-lip-circ0008828 effectively improved the cardiac function of mice

[0187] On day 21 after administration, we performed echocardiography on mice in the Nor, SuHx, and R8-Lip-circ0008828 groups. The PAT / PET ratio in the SuHx group was lower than that in the Nor group, suggesting the presence of pulmonary hypertension. In addition, the maximum velocity of pulmonary artery blood flow (PVmax) decreased, indicating possible pulmonary artery stenosis or embolism.

[0188] In contrast, the administration of R8-Lip-circ0008828 resulted in an increase in the PAT / PET ratio and PVmax, indicating that pulmonary hypertension in mice was improved on day 21 after administration. In addition, a decrease in tricuspid annular systolic excursion (TAPSE), right cardiac output (CO), and PAVTI (right ventricular stroke volume index) was observed, indicating a decrease in right ventricular function. At the same time, the administration of R8-Lip-circ0008828 resulted in an increase in PVmax, PAVTI, TAPSE, and CO in mice, indicating that circ0008828 successfully improved pulmonary hypertension-induced right ventricular dysfunction, and this effect persisted on day 21 after administration.

[0189] This series of results further emphasizes the positive effects of R8-Lip-circ0008828, especially in the treatment of pulmonary hypertension and related right ventricular function decline, and these improvements were still observed on day 21 after administration. These findings provide useful information for the treatment of pulmonary hypertension and provide strong support for further research and clinical application.

[0190] Fig.21 A. Representative images and statistical graphs of pulmonary artery hemodynamics and right ventricular function in mice with pulmonary hypertension assessed by small animal transthoracic echocardiography. Fig. 22 , B. Pulmonary artery blood flow acceleration time (PAT); Fig.23 , C. The ratio of pulmonary artery acceleration time to pulmonary artery ejection time (PAT / PET); Fig.24 , D. Maximum pulmonary artery blood flow velocity (PVmax); Fig.25 , E. Pulmonary artery velocity time integral (PAVIT); Fig.26 , F. tricuspid annular systolic excursion (TAPSE); Fig. 27 , G. Right ventricular output (CO).

[0191] (11) R8-lip-circ0008828 effectively restored the expression of the target gene

[0192] Previous studies have found that hsa_circ0008828 may play its role in PAH by targeting the hsa-miR-29b-2-5p-KCNB1 axis. Consistent with previous findings, hypoxia significantly inhibited the expression level of circ0008828, while increasing the expression level of miR-29b and significantly reducing the mRNA level of the KCNB1 gene. However, the administration of R8-Lip-circ0008828 significantly reversed this hypoxia-induced inhibition and partially restored the expression levels of miR-29b and KCNB1. These results indicate that the administration of R8-Lip-circ0008828 successfully restored the expression level of the target gene KCNB1 and exerted its therapeutic effect on PAH through the circ0008828-miR-29b-KCNB1 axis. This provides molecular mechanistic support for R8-Lip-circ0008828 as a potential PAH treatment and highlights its important role in restoring gene expression. This has important clinical and scientific value for understanding and treating PAH and related diseases.

[0193] Fig.28 , A. qPCR detection of the expression level of circ0008828; Fig.29 .qPCR was used to detect the expression level of miR-29b; Fig.30 .qPCR was used to detect the expression level of KCNB1. Fig.31 .Representative confocal images of circ0008828 (red), miR-29b (green), KCNB1 (pink purple), and DAPI (blue) in pulmonary artery sections of each group of mice; Fig.32 Fluorescence quantitative image E, Fig.33 Fluorescence quantitative image F, Fig.34 . Fluorescence quantification graph G.

[0194] (12) R8-lip-circ0008828 has no damage to other important organs of mice

[0195] R8-lip-circ0008828 was intranasally administered to normal mice for two weeks, and the main organs of the mice were stained with HE and histologically analyzed. The results showed that R8-lip-circ0008828 had no damage to other important organs of mice.

[0196] Circular RNA (hsa_circ_0008828) Gene encoding circular RNA molecule (gene ID: hsa_circ_0008828).

Claims

1. A circular RNA molecule drug for the treatment of pulmonary hypertension in mice, characterized by: The ribonucleotide circular RNA molecule hsa_circ_0008828 was cloned and purified, then modified with liposomes and administered nasally to mice. It was effectively delivered to the mouse lung tissue and had a therapeutic effect on pulmonary hypertension model mice, providing a new type of molecular drug for the clinical treatment of pulmonary hypertension.

2. The therapeutic effect of the circular RNA molecule drug according to claim 1 on mice with pulmonary hypertension is characterized by: Based on the ribonucleotide circular RNA molecule hsa_circ_0008828, the transmembrane lipid drug R8-Lip-circ0008828 was constructed, and its therapeutic effect in the mouse pulmonary hypertension model was evaluated by intranasal administration.

3. The therapeutic effect of the circular RNA molecule drug according to claim 1 or 2 on mice with pulmonary hypertension is characterized in that: The first step is to construct the liposome-modified drug R8-Lip-circ0008828, the second step is to administer R8-Lip-circ0008828 to mice intranasally, the third step is to verify the drug tissue distribution by in vivo imaging of mice, the fourth step is to establish a mouse pulmonary hypertension model, the fifth step is to evaluate the efficacy of R8-Lip-circ0008828 intranasally for the treatment of pulmonary hypertension in mice: the sixth step is mouse echocardiography, the seventh step is to determine the right ventricular hypertrophy index of mice, the eighth step is hematoxylin-eosin staining of mouse organ tissues, the ninth step is fluorescent in situ hybridization of mouse lung tissues, the tenth step is immunofluorescence of mouse lung tissues, the eleventh step is immunoblot analysis of mouse lung tissues, the twelfth step is real-time fluorescence quantitative polymerase chain reaction verification of mouse lung tissues, and the thirteenth step is biological statistical analysis.

4. The therapeutic effect of the circular RNA molecule drug according to claim 3 on mice with pulmonary hypertension is characterized in that: The first step is to construct the liposome-modified drug R8-Lip-circ0008828. Step 1-cloning and purification of hsa_circ_0008828 plasmid: constructing the expression vector of hsa_circ_0008828, and adopting the conventional plasmid cloning and purification method to perform vector transformation and plasmid extraction to obtain 5 mg of hsa_circ_0008828 expression plasmid; Step 2-Preparation of modified liposomes: ① Mix octaarginine R8 and distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide DSPE-PEG2000-Mal with chloroform and methanol at a molar ratio of 1.5:1, and then stir at room temperature for 48 hours; ② The mixture is evaporated in a vacuum rotary evaporator to obtain a precipitate, which is then redissolved in chloroform; the supernatant distearoyl phosphatidylethanolamine-polyethylene glycol 2000-octaarginine conjugate DSPE-PEG2000-R8 is obtained by filtration, and the supernatant distearoyl phosphatidylethanolamine-polyethylene glycol 2000-octaarginine conjugate DSPE-PEG2000-R8 is evaporated under reduced pressure at 40° C. and 40 revolutions per minute to form a lipid film; Step 3-Preparation of liposome film: ① Mix lecithin SPC, cholesterol Chol / distearoylphosphatidylethanolamine-polyethylene glycol 2000 DSPE-PEG2000 and distearoylphosphatidylethanolamine-polyethylene glycol 2000-octaarginine conjugate DSPE-PEG2000-R8 at a molar ratio of 59:33:3:5, and add them to anhydrous ethanol; ② The mixture was passed through a rotary evaporator to form a lipid film, and the round-bottom flask containing the lipid film was placed at 4°C for 2 hours; Step 4 - Aqueous Dispersion: Dissolve the lipid film with 3 mL of phosphate buffered saline (PBS) and place in a 60°C water bath for 0.75 h; Intermittent sonication was performed using a probe sonicator at 100 watts for 0.16 h and filtered through a 0.22 μm filter membrane to ensure uniform dispersion of the liposome vesicles; Step 5 - Packaging of hsa_circ_0008828: 5 mg of purified hsa_circ_0008828 plasmid was mixed with blank liposomes and then incubated at 4 °C for 2 h to prepare liposome R8-Lip-circ0008828 containing 2 mg / mL hsa_circ_0008828; Step 6 - Analysis and Characterization: ① Use the Malvern high-sensitivity nanoparticle size analyzer to detect the particle size and polymer dispersibility index of liposome nanoparticles; ② Use laser particle size distribution and zeta potential instrument to detect the zeta potential of nanoparticles; ③ Use agarose gel electrophoresis to detect the encapsulation of hsa_circ_0008828; ④ Use a transmission electron microscope to observe the morphology of the sample and take photos; ⑤ Use mouse red blood cells to detect the biosafety of R8-Lip-circ0008828 liposome nanoparticles; In steps 1-5, the lipid-modified R8-Lip-circ0008828 was constructed as a hsa_circ_0008828 circular RNA therapeutic drug for pulmonary arterial hypertension.

5. The therapeutic effect of the circular RNA molecule drug according to claim 3 on mice with pulmonary hypertension is characterized in that: The second step R8-Lip-circ0008828 was intranasally administered to mice. Step 1: 25 mg of 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindole dicarbon cyanine perchlorate was dissolved in 5.21 ml of anhydrous ethanol to obtain 5 mmol of 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindole dicarbon cyanine perchlorate DID storage solution. Phosphate buffered saline PBS was diluted 1000 times to obtain 5 micromolar 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindole dicarbon cyanine perchlorate DID working solution; Step 2: The prepared R8-Lip-circ0008828 lipid-modified body and the control vector R8-Lip-PLC5 lipid-modified body were mixed with an equal volume of DID working solution and incubated at 4°C for 2 hours; Step 3, the mice were randomly divided into 3 groups: free DID, R8-Lip-PLC5-DID, and R8-Lip-circ0008828-DID; each group of mice was anesthetized with tribromoethanol and then intranasally administered; specific administration method: the mice were anesthetized with 4% mass concentration of tribromoethanol, the mice were picked up and their bodies were placed in a supine position, and tilted at an angle of 45°, and R8-Lip-circ0008828 was slowly administered drop by drop with a sterile pipette to the outside of the mouse nostrils, 0.5 mg / mL, and the mice were inhaled with breathing, and 50 microliters were dripped into each nostril; after administration, the mice were put back into the cage and allowed to recover from anesthesia on their own; The third step of in vivo imaging of mice to verify the tissue distribution of the drug, step 1, anesthetizing the mice 1 hour, 6 hours, 12 hours, and 24 hours after administration, placing them in a supine position in a small animal in vivo imaging instrument, selecting chemiluminescence, setting the excitation wavelength to 640 nanometers, taking pictures and recording; Step 2: 24 hours after the injection, the mice were killed, and the main organs of the mice, including brain, heart, liver, spleen, lung, and kidney, were taken and placed in a small animal in vivo imaging device to capture in vitro fluorescence.

6. The therapeutic effect of the circular RNA molecule drug according to claim 3 on mice with pulmonary hypertension is characterized by: The fourth step is to establish a mouse pulmonary hypertension treatment model. Modeling was performed using specific pathogen-free grade C57BL / 6J male mice 20-22 g according to the following steps: Step 1, adaptive feeding: The mice were adaptively fed for 7 days to ensure their health status and body weight stability; Step 2, grouping: mice were randomly divided into two groups; Control group: fed in a normoxic environment, provided with adequate food and water; Pulmonary hypertension model group: The pulmonary hypertension mouse model was established by subcutaneous injection of semasanib at a concentration of 20 mg / kg once a week, combined with hypoxia and 10% oxygen feeding for 21 days; Step 3, anesthesia and hemodynamic and echocardiographic analysis: On the 14th and 21st days of the experiment, the mice were anesthetized with 4% mass concentration of tribromoethanol, and hemodynamic and echocardiographic analyses were performed to evaluate cardiovascular function; Step 4, thoracotomy and organ collection: The mice were subjected to thoracotomy and perfused with phosphate buffered saline (PBS), and then their organs were collected for subsequent experiments.

7. The therapeutic effect of the circular RNA molecule drug according to claim 3 on mice with pulmonary hypertension is characterized by: The fifth step of the intranasal administration of R8-Lip-circ0008828 to treat the pulmonary hypertension mouse model; R8-Lip-eirc0008828 treated pulmonary hypertension model group: The pulmonary hypertension mouse model was established according to step 4, but R8-Lip-circ0008828, 0.5 mg / mL, was intranasally administered daily from the 7th day to the 21st day according to step 3, and the mice were raised in a hypoxic environment for 21 days; the mice were intranasally administered with phosphate buffered saline (PBS) as the drug control group; The sixth step of the mouse echocardiogram uses a small animal ultrasound instrument to record a transthoracic echocardiogram to detect the cardiac function and hemodynamics of the model mouse. The MS400 probe is used to examine small rodents to obtain echocardiographic examination images of the mouse pulmonary artery and right ventricle at a stable heart rate; In the seventh step, the right ventricular hypertrophy index of mice is measured. The heart is dissected and the right ventricular hypertrophy is calculated by the ratio of the right ventricular weight to the right ventricular free wall weight plus the left ventricular septum weight RV / (LV+S), so as to characterize the degree of right ventricular hypertrophy.

8. The therapeutic effect of the circular RNA molecule drug according to claim 3 on mice with pulmonary hypertension is characterized by: In the eighth step of hematoxylin-eosin staining, the mouse lung tissue is immersed in 4% paraformaldehyde for 48 hours and then dehydrated with gradient alcohol, embedded in wax blocks after being transparent in xylene, and then sliced ​​into 5-6 micron thickness, the slices of embedded mouse lung tissue are dewaxed in xylene, rehydrated with gradient alcohol, and washed with distilled water; hematoxylin staining for 0.08 hours, differentiated with differentiation solution for 2-3 seconds, rinsed with tap water to return to blue, and eosin staining for 0.02 hours; finally, dehydrated with gradient alcohol, transparentized with xylene, sealed with neutral gum, and the reconstruction of pulmonary blood vessels was observed and photographed under a light microscope; The ninth step of fluorescent in situ hybridization is to remove the paraffin-embedded lung sections of mice in xylene, and then rehydrate them with gradient ethanol and water; add proteinase K to digest the lung sections, incubate at 37°C for 0.33 hours, wash the slides with phosphate buffered saline PBS, add denaturing solution, and denature at 78°C for 0.13 hours; add the hsa_circ_0008828 and miR-29b probe mixture to the lung tissue, incubate at 37°C overnight; discard the hybridization solution, wash three times with phosphate buffered saline PBS, and then drop KCNB1 rabbit antibody on the tissue, incubate at 4°C in the dark overnight. Add horseradish peroxidase-labeled anti-rabbit antibody and incubate for 1 hour, wash with phosphate buffered saline PBS, and then drop 4′,6-diamidino-2-phenylindole and incubate for 0-17 hours; add anti-fluorescence quenching sealing agent, cover with a cover glass, seal with nail polish, and detect with a fluorescence microscope.

9. The therapeutic effect of the circular RNA molecule drug according to claim 3 on mice with pulmonary hypertension is characterized in that: The tenth step of immunofluorescence is to dewax and rehydrate the paraffin-embedded lung sections of mice, boil the slides with 0.01 mol ethylenediaminetetraacetic acid antigen repair solution at 98°C for 0.17 hours to perform antigen repair; drip α-smooth muscle actin primary antibody on the tissue, incubate at 4°C in the dark overnight; add horseradish peroxidase HRP-labeled secondary antibody and incubate for 1 hour, drip DPAI and incubate in the dark for 0.17 hours, wash with PBS, add anti-fluorescence quenching sealing agent, cover with a cover slip, seal with nail polish, and detect with a fluorescence microscope; In the eleventh step of immunoblotting analysis, mouse lung tissue was lysed on ice using 1 mL of protein lysis buffer containing 1% phenylmethylsulfonyl fluoride, total protein was extracted, quantified using a BCA protein concentration assay kit, and then the protein was separated by electrophoresis in an SDS-PAGE gel, and the protein was transferred to a polyvinylidene fluoride membrane using a transfer apparatus, and the PVDF membrane was blocked with a 5% mass concentration skim milk blocking solution at room temperature for 2 hours, a primary antibody was added, and the membrane was incubated overnight at 4°C. After washing the membrane, an HRP-labeled goat anti-rabbit immunoglobulin G secondary antibody was added and incubated at room temperature for 1 hour; an ECL enhanced chemiluminescence colorimetric reagent was added dropwise to the PVDF membrane, and finally a gel imager was used for detection and analysis and photographing and recording.

10. The therapeutic effect of the circular RNA molecule drug according to claim 3 on mice with pulmonary hypertension is characterized in that: In the twelfth step, the real-time fluorescent quantitative polymerase chain reaction was used to extract total RNA from mouse lung tissue, and the RNA was reversed into cDNA using a complementary DNA reverse transcription kit, and primers were designed to amplify hsa_circ_0008828 and KCNB1, respectively, and β-actin was amplified as a control; In the thirteenth step of statistical analysis, data analysis was performed using unpaired t-test for two-group comparison and one-way analysis of variance, and the minimum P value of 0.05 was selected as the significance level.

Images