MiRNA-181a-5p and application of miRNA-181a-5p inhibitor in medicine for treating lower limb arteriosclerosis obliterans

Abstract

The invention relates to the technical field of medicines, in particular to miRNA-181a-5p and application of an inhibitor of miRNA-181a-5p in a medicine for treating lower limb arteriosclerosis obliterans. The research on disease pathogenesis finds that miRNA-181a-5p is particularly important in the occurrence and development process of lower limb arteriosclerosis obliterans, and the miRNA-181a-5p inhibitor is adopted to treat vascular endothelial cells, so that the functions of the vascular endothelial cells can be improved, including improvement of migration and tubular formation of the vascular endothelial cells; the proliferation function is improved; the ROS generation is reduced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of miRNA-181a-5p and its inhibitors in the treatment of lower extremity arteriosclerosis obliterans. Background Technology

[0002] Arteriosclerosis obliterans (ASO) is a disease caused by atherosclerotic plaques leading to narrowing or blockage of arteries, affecting blood supply to the lower extremities. Symptoms, from mild to severe, include intermittent claudication, rest pain, and gangrene. End-stage patients require complex surgical treatment, have poor prognosis, and face a heavy burden on patients and society. The continuous rise in cardiovascular risk factors such as hypertension, dyslipidemia, diabetes, and smoking is the main reason for the increasing incidence of ASO among residents. Therefore, the prevention and treatment of ASO should focus on controlling risk factors. Clinical research should be emphasized to develop relevant guidelines to provide patients with standardized drug treatment, including antiplatelet therapy, lipid-lowering drugs, and control of blood sugar and blood pressure, to slow the progression of atherosclerosis, improve lower extremity ischemia symptoms as much as possible, reduce the occurrence of cardiovascular and cerebrovascular events, and ultimately improve patient survival rates. Currently, there is a lack of effective drug targets for the clinical treatment of lower extremity arterial occlusive disease. Therefore, research is needed at different molecular levels and from multiple dimensions, such as proteomics and metabolomics, to develop more effective and specific biomarkers and drug targets, providing new directions for the clinical treatment of lower extremity arterial occlusive disease.

[0003] The main treatments for lower extremity arteriosclerosis obliterans currently include drug therapy and surgical and interventional treatments. Drug therapy includes: antiplatelet drugs such as aspirin and clopidogrel, which inhibit platelet aggregation and prevent thrombosis; vasodilators such as cilostazol and beraprost, which dilate blood vessels and improve blood supply to the lower extremities; lipid-lowering drugs such as statins (e.g., atorvastatin and rosuvastatin) to stabilize plaques and slow vascular lesions; and analgesics used when necessary to relieve resting pain. Overall, drug therapy can slow disease progression, but its effectiveness in improving or even reversing the disease is not ideal. Interventional procedures such as balloon angioplasty or stent implantation use catheters to deliver balloons or stents to the narrowed or occluded sites to dilate blood vessels and restore blood flow. These procedures can improve vascular patency in the short term, but long-term effects are poor, and restenosis and occlusion may occur. Therefore, further research is needed on lower extremity arteriosclerosis obliterans to identify the main pathogenesis of the disease, block its progression at its root, and improve lower extremity ischemia and hypoxia by promoting angiogenesis. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an application of miRNA-181a-5p and its inhibitor in the treatment of lower extremity arteriosclerosis obliterans.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the application of miRNA-181a-5p in the preparation of a drug for treating lower extremity arteriosclerosis obliterans.

[0006] Another technical solution adopted in this invention is the application of an inhibitor of miRNA-181a-5p in the preparation of a drug for treating lower extremity arteriosclerosis obliterans.

[0007] The beneficial effects of this invention are as follows: By studying the pathogenesis of the disease, this invention has found that miRNA-181a-5p is particularly important in the occurrence and development of lower extremity arteriosclerosis obliterans. Treatment of vascular endothelial cells with miRNA-181a-5p inhibitors can improve the function of vascular endothelial cells, including improving their migration and tubular formation; improving their proliferative function and reducing the production of ROS (reactive oxygen species). Attached Figure Description

[0008] Figure 1 This is a graph showing the tubular formation ability test of endothelial cells in each group in Embodiment 1 of the present invention; Figure 2 for Figure 1 Statistical charts; Figure 3 This is a graph showing the effect of blood perfusion in the lower limbs of mice in each group in Example 2 of the present invention; Figure 4 This is a statistical chart showing the blood perfusion status of the lower limbs of mice in each group in Example 2 of the present invention; Figure 5 These are CT images of mice in each group in Example 2 of the present invention; Figure 6 The images show the CT statistics of mice in each group in Example 2 of this invention. Detailed Implementation

[0009] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0010] Application of miRNA-181a-5p in the preparation of drugs for the treatment of lower extremity arteriosclerosis obliterans.

[0011] As can be seen from the above description, the beneficial effects of the present invention are as follows: by studying the pathogenesis of the disease, the present invention has found that miRNA-181a-5p is particularly important in the occurrence and development of lower extremity arteriosclerosis obliterans.

[0012] miRNAs play a crucial role in regulating angiogenesis, and they can further modulate angiogenesis by influencing autophagy. Normal high-density lipoprotein (nHDL) induces angiogenesis by inhibiting miR-181a-5p, but patients often express this protein excessively, leading to suppressed angiogenesis. Therefore, miRNA-181a-5p could be used to treat arteriosclerosis obliterans of the extremities.

[0013] Furthermore, miRNA-181a-5p is used to regulate angiogenesis.

[0014] Furthermore, a product was developed targeting miRNA-181a-5p to predict the prognosis of lower extremity arteriosclerosis obliterans.

[0015] Another technical solution adopted in this invention is the application of an inhibitor of miRNA-181a-5p in the preparation of a drug for treating lower extremity arteriosclerosis obliterans.

[0016] As described above, this invention utilizes miRNA-181a-5p inhibitors to treat vascular endothelial cells, which can improve vascular endothelial cell function, including improving migration and tubular formation; improving proliferative function; and reducing ROS production. In vivo animal experiments have shown that miRNA-181a-5p inhibitor treatment can improve lower limb arterial occlusion and promote angiogenesis and remodeling. By improving systemic angiogenesis capacity, it can achieve long-term improvement of lower limb ischemia and hypoxia, and ultimately, a complete cure for lower limb vascular occlusive disease.

[0017] Furthermore, inhibitors of miRNA-181a-5p are used to promote angiogenesis.

[0018] Furthermore, inhibitors of miRNA-181a-5p are used to promote vascular remodeling.

[0019] Furthermore, inhibitors of miRNA-181a-5p are used to improve the function of vascular endothelial cells.

[0020] Furthermore, inhibitors of miRNA-181a-5p are used to improve vascular endothelial cell migration and tubular formation.

[0021] Furthermore, inhibitors of miRNA-181a-5p are used to improve the proliferative function of vascular endothelial cells.

[0022] Furthermore, inhibitors of miRNA-181a-5p are used to improve ROS production in vascular endothelial cells.

[0023] Embodiment 1 of the present invention is a cell experiment, the specific steps of which are as follows: 1. Cell culture: Vascular endothelial cells were cultured and seeded in six-well plates. When the cell density reached approximately 50%–60%, they were diluted to 50 nM with miR-181a-5p mimics and inhibitor, respectively, and transfected according to the supplier's manual. The transfection steps are as follows: Add 100 μL of optimized medium (Opti-MEM) to a sterile EP tube, then add 3 μL of lipofectamine RNAiMAX Regent transfection reagent, and gently vortex to mix. Prepare three new EP tubes, add 100 μL of optimized medium, then add 2 μL of mimics, 2 μL of inhibitor (50 nM), and 2 μL of negative control (50 nM) to each tube. Mix by pipetting several times with 100 μL of the mixture, then combine each mixture with the transfection reagent mixture in the EP tube. Mix by pipetting several times with 100 μL of the mixture, place them on the EP tube rack in the cell culture stage, let stand for 15 min, then add optimized medium to 1000 μL, mix well, and set aside. Remove the culture medium from the 6-well plate, wash it once with warmed PBS (phosphate-buffered saline), then wash away any residual PBS. Add 1 mL of the prepared solution to the corresponding well of the 6-well plate, return it to the incubator and continue culturing. After 12 hours, replace the culture medium with complete medium. (For transfection of mimics, the transfection time should not be too long, preferably 7 hours, otherwise too many cells will die and the cells will not reach confluence.) Continue culturing in the incubator until the cell density reaches about 90% within 48 hours of transfection, then it can be used for subsequent experiments.

[0024] 2. After transfecting cells with miR-181a-5p mimics and inhibitor as described above, once the cell density in a 6-well plate or culture dish is >90%, discard the original complete culture medium, add starvation medium to the 6-well plate and starve overnight, then add starvation medium containing 100 μg / mL nHDL or dHDL, incubate for 24 h, and then process or collect the samples. Set up a blank group as a control.

[0025] 3. Cell tube formation assay to verify endothelial cell regeneration function. 3.1 Processing of Matrix Gel The night before the experiment, transfer the matrix gel from the -20°C freezer to ice and thaw it in a 4°C freezer, taking care not to expose it to room temperature for an extended period. Place 200 μL of sterilized pipette tips in a sealed plastic bag and freeze them in a -20°C freezer for pre-cooling before use.

[0026] On the day of the experiment: Place a 96-well plate, pre-chilled pipette tips, and dissolved matrix gel in a small foam box filled with ice. Place the foam box in a clean bench. Using the pre-chilled pipette tips, pipette 60 μL of matrix gel into the 96-well plate, and set up the groups as needed. Care should be taken to avoid air bubbles during the pipette aspiration and addition of the matrix gel to the wells. Then, gently shake the 96-well plate to ensure even distribution of the matrix gel. During aspiration, the pipette can be set to speed 2 for the first aspiration, but only speed 1 for subsequent layering. This effectively avoids the aspiration and introduction of gas during layering. After layering, incubate the 96-well plate in a cell culture incubator for 30 minutes to allow the matrix gel to solidify.

[0027] 3.2 Digest and count cells HUVECs (human umbilical vein endothelial cells) were cultured in 6-well plates according to the cell culture method described above. After overexpression treatment of the cells as described above, the culture medium was discarded, and 2 mL of PBS was added to each well to wash the cells twice. Then, 0.5 mL of trypsin was added to each well for about 40 seconds. The cell plate was gently tapped, and the adherent cells were observed to be suspended under a microscope. Then, an equal volume of complete culture medium was added to neutralize the trypsin, and the cells were gently pipetted through the wells to digest them and collect them into clean, sterile EP tubes. The cells were centrifuged at 1000 rpm for 5 minutes at room temperature, and the supernatant was discarded to obtain the cell pellet. Then, about 1 mL of starvation medium was added to resuspend the cells, and 10 μL of each tube was added to a cell counting plate for counting.

[0028] Based on the cell count results, the cell suspension was diluted with starvation medium to uniformly adjust the density to 1.2 × 10⁻⁶. 5 Cells were added at a rate of 100 μg / mL, with the corresponding nHDL (100 μg / mL), dHDL (100 μg / mL), and VEGF (50 ng / mL) added according to the previously grouped cells. A control group was also included. Then, 100 μL of the cell suspension with the added stimulating drugs was added to each solidified matrix gel. The 96-well plate with the added cells was gently shaken to mix, and then incubated in a cell culture incubator for 6–12 h.

[0029] 3.3 Records After adding the drug, the cell plate was placed in a live-cell imaging system for 7 hours at fixed times and locations. The number of vascular strips formed in each group was counted and recorded using ImageJ software. The results are shown below. Figure 1 and 2 .

[0030] like Figure 1 and 2As shown, compared with the Mimic-NC group, HDL in normal individuals significantly promoted endothelial cell tube formation, while HDL in patients did not promote endothelial cell tube formation; miR-181a-5p inhibitor significantly improved endothelial cell tube formation in the dHDL group, while the VEGF group served as a positive control.

[0031] Embodiment 2 of the present invention is an animal experiment, the specific steps of which are as follows: 1. C57:C57BL / 6 mice were selected and divided into three groups of 5 mice each. One group (ND) was fed a normal diet, consisting of 56g of basal feed (10% of calories) per day, with free access to water, for eight weeks. The other two groups were fed a high-fat diet (WD and iRNA-181a-5p inhibitor), consisting of 56g of high-fat feed (50% of calories) per day, with free access to water, for eight weeks.

[0032] 2. Establishment of a lower limb ischemia model Mice were anesthetized with an intraperitoneal injection of 50 mg / kg pentobarbital and fixed in a supine position on a foam board. Hair was trimmed from the left hind leg, thigh, left groin, and left abdomen. The skin was disinfected with 75% alcohol. The left groin area was chosen as the surgical approach. The skin was cut approximately 1 cm open with sterile scissors. The fascia layer was cut open, and the femoral artery sheath was bluntly dissected under a stereoscope. The femoral artery was then dissected proximally on the main trunk of the femoral artery, approximately 5 mm from the branch of the deep femoral artery, taking care not to damage the accompanying femoral nerve and vein. Two 10-0 silk sutures were used to double-ligate both ends of the femoral artery, and the femoral artery was transected between the two ligation points. The surgical field was disinfected with povidone-iodine, and then the mouse skin wound was continuously sutured with 4-0 silk sutures.

[0033] Mice in the iRNA-181a-5p inhibitor group were injected with miR-181a-5p inhibitor. On the day the lower limb ischemia model was established and on day 15, the blood perfusion of the mice in each group was monitored using laser Doppler flowmeter. After day 15, the mice were euthanized by overdose anesthesia. Blood samples were collected by intracardiac injection in the right atrium. A small incision was made in the right atrial appendage, and residual blood was flushed out through injection of saline into the left ventricle. A catheter was placed through the abdominal aorta, and the proximal end was ligated. 4% formaldehyde was injected into the selected end to fix the tissue. Contrast agent prepared according to the manufacturer's instructions was injected through the catheter. After overnight incubation at four o'clock, decalcification was performed for three weeks using decalcification solution. Small animal CT angiography was then used to observe microvascular density in the lower limbs. Gastrocnemius muscle samples from some mice in the ischemic lower limbs were harvested and stored at -80°C or used directly in experiments.

[0034] 3. Measurement of blood perfusion After establishing the mouse hind limb ischemia model as described above, laser Doppler ultrasound was immediately used to detect the blood perfusion of the mouse's hind limbs. The specific steps are as follows: First, turn on the laser Doppler instrument, calibrate it according to the manual, name the new program, and set the scanning parameters as follows: autofocus, 50 scan images, 5 frames / second, and average 5 images. After preparation, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). If the mice were still anesthetized after surgery, further anesthesia was not necessary. The mice were placed prone on a black mouse pad, with the palms and fingers of both hind limbs within the scanning area. Double-sided tape was used to attach the backs of the mice's hands to the mouse pad, and the palms and fingers were appropriately separated with tweezers. The scan was started, and the blood perfusion of the mouse's hind limbs was recorded and the blood perfusion volume was calculated. The above operation was repeated 15 days after surgery to record the recovery of postoperative blood flow.

[0035] 4. CT angiography Fifteen days post-surgery, after blood flow testing, the mice remained anesthetized. If they awoke, an appropriate dose of sodium pentobarbital was administered to anesthetize them, taking care not to overdose and cause death. After successful anesthesia, the mice were fixed in a supine position on a board. The abdominal skin was cut open, the peritoneum opened, and the abdominal cavity was accessed. The intestinal tissue was appropriately dissected to expose the abdominal aorta. Two 10-gauge silk sutures were prepared. A small incision was made in the abdominal aorta using microsurgical scissors, and a PE10 catheter was quickly inserted, with the distal end facing away from the head and towards the feet. The lower end of the suture was tied to secure it, taking care not to apply too much force to avoid collapse and occlusion of the catheter wall. The thoracic cavity was opened, and 1 ml of blood was drawn from the right atrium using a syringe. Residual blood was then slowly perfused with physiological saline through the inserted catheter. Formaldehyde was then injected through the catheter, and after approximately 30 minutes of fixation, a contrast agent was prepared according to the manufacturer's instructions. A 5 ml syringe was used to inject the contrast agent under pressure through the catheter. At this point, the contrast agent was visible as it injected into the small arteries and appeared yellow. Injection was performed evenly, avoiding air bubbles. The animal was left to harden overnight at 4°C, and then soaked in a decalcification solution for three weeks for decalcification treatment. Small animal CT imaging was then performed.

[0036] 5. Result Prompt: 5.1 Inhibition of miRNA-181a-5p can increase microvascular density in the ischemic lower limbs of hypercholesterolemic mice. On the day of surgery and 15 days post-surgery, laser Doppler flowmeter was used to assess blood perfusion in the hind limbs of mice. Figure 3 and 4 In this study, ND represented a normal diet, WD represented a high-fat diet, and the miR-181a-5p inhibitor group received a high-fat diet and was injected with miR-181a-5p inhibitor; NC represented an empty vector control virus. * vs. C57+ND, # vs. LDLr- / - + WD+NC, p<0.05, n=8.

[0037] The results of tests on the day of surgery showed that, in all groups, after ligation of the femoral artery, the blood flow on the affected side decreased by more than 80% compared to the healthy side, proving the model was successful. Comparison of blood flow recovery 15 days post-surgery and on the day of surgery showed that in the ischemic lower limb, normal C57BL / 6 mice (C57+ND+NC) had normal blood perfusion restored on day 15 post-surgery, but the blood perfusion of hypercholesterolemia mice injected with the control virus group (LDLr- / -+WD+NC) was significantly reduced compared to normal C57BL / 6 mice (C57+ND+NC). In hypercholesterolemia mice (LDLr- / -+WD), the blood perfusion of the ischemic lower limb injected with the miRNA-181a-5p inhibitor was significantly higher than that of the control virus group (LDLr- / -+WD+NC), and essentially reached the level of C57BL / 6 mice (C57+ND+NC).

[0038] 5.2 Inhibition of miRNA-181a-5p can increase angiogenesis in the ischemic lower limbs of hypercholesterolemic mice. Fifteen days post-surgery, after angiography and decalcification fixation of the specimens, small animal CT scans were used to examine the microvessels in the mouse hind limbs. Figure 5 and 6 The ND group consisted of a normal diet, the WD group consisted of a high-fat diet, the miR-181a-5p inhibitor group consisted of a high-fat diet and injection of miR-181a-5p inhibitor, and the NC group consisted of an empty vector plasmid. * vs. C57+ND, # vs. LDLr- / - +WD+NC, p<0.05, n=8.

[0039] CT results showed that in normal C57BL / 6 mice (C57+ND+NC), microvessel density in the ischemic limb recovered to the level of the contralateral non-ischemic limb on day 15 post-surgery. However, the microvessel density in hypercholesterolemic mice (LDLr- / -+WD+NC) was significantly reduced compared to normal C57BL / 6 mice (C57+ND+NC) and the contralateral non-ischemic limb. In hypercholesterolemic mice (LDLr- / -+WD), the microvessel density in the ischemic limb injected with miRNA-181a-5p inhibitor (LDLr- / -+WD+miRNA-181a-5p inbitor) was significantly increased compared to the control group (LDLr- / -+WD+NC), reaching the level of C57BL / 6 mice (C57+ND+NC) and the contralateral non-ischemic limb. Figure 3 ).

[0040] Embodiment 3 of the present invention is: the application of miRNA-181a-5p in the preparation of a drug for treating lower extremity arteriosclerosis obliterans. miRNA-181a-5p regulates angiogenesis, and products for predicting the prognosis of lower extremity arteriosclerosis obliterans can be prepared by targeting miRNA-181a-5p.

[0041] Example 4 of the present invention is the application of an inhibitor of miRNA-181a-5p in the preparation of a drug for treating lower extremity arteriosclerosis obliterans. The inhibitor promotes angiogenesis and remodeling, improves cell migration and tubular formation of vascular endothelial cells, and improves the proliferative function of vascular endothelial cells and ROS production.

[0042] In summary, the application of miRNA-181a-5p and its inhibitors provided by this invention in the preparation of therapeutic drugs for lower extremity arteriosclerosis obliterans demonstrates the inhibitory effect of miR-181a-5p on angiogenesis in patients using cell transfection technology in vitro experiments. Similarly, by inhibiting miR-181a-5p expression, a significant recovery of endothelial angiogenesis function can be observed. In vivo animal experiments then show that treatment with miRNA-181a-5p inhibitors can improve lower extremity arterial occlusion and promote angiogenesis and remodeling. By improving systemic angiogenesis capacity, it can achieve long-term improvement of lower extremity ischemia and hypoxia, thereby fundamentally curing lower extremity vascular occlusive disease.

[0043] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. Use of miRNA-181a-5p in the preparation of a medicament for treating arteriosclerosis obliterans of lower extremities.

2. Use according to claim 1, characterized in that, The miRNA-181a-5p is used for regulating angiogenesis.

3. Use according to claim 1, characterized in that, A product for predicting the prognosis of arteriosclerosis obliterans of lower extremities is prepared by taking miRNA-181a-5p as a target.

4. Use of an inhibitor of miRNA-181a-5p in the preparation of a medicament for treating arteriosclerosis obliterans of lower extremities.

5. Use according to claim 4, characterized in that, The inhibitor is used for promoting angiogenesis.

6. Use according to claim 4, characterized in that, The inhibitor is used for promoting vascular reconstruction.

7. Use according to claim 4, characterized in that, The inhibitor is used for improving the function of vascular endothelial cells.

8. Use according to claim 7, characterized in that, The inhibitor is used for improving the migration and tube formation of vascular endothelial cells.

9. Use according to claim 7, characterized in that, The inhibitor is used for improving the proliferation function of vascular endothelial cells.

10. Use according to claim 7, characterized in that, The inhibitor is used for improving the ROS production of vascular endothelial cells.

Images