Application of NCOA3 polyQ structural domain as target spot in preparation of medicine for relieving lower limb ischemic diseases

Abstract

The invention provides application of an NCOA3 polyQ structural domain as a target spot in preparation of a medicine for relieving lower limb ischemic diseases, relates to the technical field of biomedicine, and aims to solve the problems that in the prior art, blood flow reperfusion recovery of the lower limb ischemic diseases is poor, collateral vessels are insufficient in formation, and safe and effective targeted intervention means are lacked. By constructing a mouse lower limb ischemia model, it is proved that blood flow recovery and collateral angiogenesis after ischemia are remarkably inhibited by Nco3polyQ structural domain deletion: compared with a WT mouse, postoperative blood flow perfusion of an Nco3Q / Q mouse is recovered slowly, and the blood flow of the affected side is only recovered by 60% on the 21st day after the operation; the CD31 positive region of the gastrocnemius muscle tissue on the 14th day after the operation is obviously reduced, and the mRNA expression of the blood vessel marker gene Pecam1 is reduced. On the basis, an intervention strategy aiming at the NCOA3 polyQ structural domain is used for preparing the medicine for relieving the lower limb ischemic disease, and a new treatment strategy and a potential target are provided for the ischemic limb disease.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate lower limb ischemic diseases. Background Technology

[0002] Lower limb ischemic diseases are a group of diseases characterized by insufficient perfusion of lower limb tissues due to peripheral arterial stenosis or occlusion. Symptoms may include intermittent claudication, rest pain, coldness and numbness in the limbs, skin trophic disorders, and ulceration and gangrene. In severe cases, it can progress to critical limb ischemia and face the risk of amputation. Functional recovery after lower limb ischemia depends on the revascularization of ischemic tissues, involving multiple processes such as collateral vessel formation, capillary angiogenesis, and vascular remodeling. Due to the persistent presence of factors such as inflammation, oxidative stress, metabolic abnormalities, and endothelial dysfunction in the ischemic microenvironment, some individuals experience a decline in angiogenesis capacity after ischemia, leading to slow blood flow recovery, tissue necrosis, and an increased risk of recurrence.

[0003] Current clinical treatment for lower extremity ischemic diseases primarily focuses on risk factor control, drug therapy, and interventional / surgical revascularization. However, challenges remain, including significant individual variability in efficacy, restenosis or recurrence, and poor distal vascular conditions or unsuitability for revascularization in some patients. Particularly in promoting the establishment of collateral circulation and long-term stable perfusion recovery after ischemia, safe, effective, and sustainable targeted interventions are still lacking. Therefore, identifying new key regulatory targets and developing translational treatment strategies based on them is a crucial research direction for improving the prognosis of lower extremity ischemic diseases. Summary of the Invention

[0004] The purpose of this application is to address the problems of poor blood flow reperfusion recovery, insufficient collateral vessel formation, and lack of effective targeted intervention methods in the prior art for lower limb ischemic diseases.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] Application of NCOA3 polyQ domain as a target in the preparation of drugs to alleviate lower limb ischemic diseases.

[0007] Preferably, the drug is used to inhibit the function of the NCOA3 polyQ domain or to block angiogenesis in the street.

[0008] Preferably, the drug comprises at least one of a nucleic acid intervention agent targeting the NCOOA3 polyQ domain, a gene editing intervention system, and a small molecule compound.

[0009] Preferably, the lower limb ischemic diseases include peripheral artery disease-related lower limb ischemia, ischemic limb hypoperfusion, and post-ischemic collateral circulation disorder.

[0010] Preferably, the drug also includes other medically acceptable adjuvants.

[0011] Preferably, the drug is one of the following: injection, tablet, capsule, granule, lyophilized powder for injection, ointment, or gel.

[0012] This application also provides a drug for relieving lower limb ischemic diseases, wherein the drug inhibits the function of the NCOA3 polyQ domain or blocks angiogenesis in the street.

[0013] Compared with the prior art, this application has the following beneficial effects:

[0014] This application reveals for the first time the direct association between the NCOA3 polyQ domain and the process of blood flow restoration and collateral vessel formation after lower limb ischemia, through Ncoa3 △Q / △Q In vivo functional loss experiments in mice demonstrated that the deletion of the Ncoa3 polyQ domain significantly inhibited the recovery of blood flow perfusion after lower limb ischemia and reduced the levels of vascular markers in ischemic tissue, providing a new direction for developing intervention strategies targeting the Ncoa3 polyQ domain for lower limb ischemic diseases. Specifically, in a mouse lower limb ischemia model, blood flow in WT mice essentially returned to normal levels on day 21 post-surgery, while the Ncoa3 domain... △Q / △Q In mice, blood flow on the affected side recovered by only about 60%, suggesting that the polyQ domain plays an important role in blood flow compensation after ischemia; at the tissue level, Ncoa3 was observed on postoperative day 14. △Q / △Q The CD31-positive region in the mouse gastrocnemius muscle was significantly reduced; at the molecular level, Pecam1 mRNA expression was significantly decreased. These results collectively support the NCOA3 polyQ domain as an important structural basis for regulating post-ischemic angiogenesis and collateral circulation formation, and possess potential clinical value as a drug development target. Attached Figure Description

[0015] Figure 1 The images shown are laser speckle flowmeter images (A) and lower limb average perfusion ratio statistics (B) at different time points after surgery in a lower limb ischemia model according to an embodiment of the present invention, wherein the average perfusion ratio = blood flow on the affected side / blood flow on the healthy side × 100%.

[0016] Figure 2 This is an immunohistochemical image of the gastrocnemius muscle tissue on the 14th day after surgery, according to one embodiment of the present invention.

[0017] Figure 3 This is a diagram showing the results of qPCR detection of Pecam1 mRNA expression in gastrocnemius muscle tissue on postoperative day 14, according to one embodiment of the present invention. Detailed Implementation

[0018] This application provides the use of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate lower limb ischemic diseases, wherein the drugs are used to inhibit the function of the NCOA3 polyQ domain or block angiogenesis in the lower limbs.

[0019] The lower limb ischemic diseases include peripheral artery disease-related lower limb ischemia, insufficient blood perfusion in ischemic limbs, and impaired collateral circulation formation after ischemia.

[0020] In one embodiment, the drug includes at least one of a nucleic acid intervention agent targeting the NCOA3 polyQ domain, a gene editing intervention system, and a small molecule compound.

[0021] The drug also includes other medically acceptable adjuvants.

[0022] The drug is one of the following: injection, tablet, capsule, granule, lyophilized powder for injection, ointment, or gel.

[0023] In addition, this application also provides a drug for relieving lower limb ischemic diseases, wherein the drug inhibits the function of the NCOA3 polyQ domain or blocks angiogenesis in the street.

[0024] The above content will be explained in the following specific verification experiment: I. Experimental Materials

[0025]

[0026] II. Experimental Procedure

[0027] Example 1: Construction of a mouse lower limb ischemia model and dynamic monitoring of blood flow

[0028] (1) Preoperative preparation: Weigh the mice one day before the operation, anesthetize the mice with 0.01 mL / g 1% sodium pentobarbital, place the anesthetized mice in a supine position in front of the operating table, remove the hair on the lower limbs with an electric shaver, and then completely remove the hair with depilatory cream. Record the blood flow of the mice in both lower limbs before the operation with a blood flow meter, and then place them in an incubator to wait for them to wake up.

[0029] (2) On the day of the operation, the anesthetized mice were transferred to a heating pad covered with a sterile surgical drape, the lower limbs were fixed with tape, the surgical area was wiped repeatedly with iodine solution at least 3 times, and then a perforated drape was laid;

[0030] (3) Under the anatomical microscope, adjust the field of view to determine the surgical area, and use curved forceps and surgical scissors to make a skin incision of about 1 cm from the knee to the inner thigh;

[0031] (4) Gently scrape away the subcutaneous fat and connective tissue around the thigh muscles with a cotton swab moistened with PBS to expose the muscles;

[0032] (5) Use an electrocautery pen to make a transverse incision in the subcutaneous adipose tissue to expose the femoral artery below;

[0033] (6) Use a paperclip-made traction device to pull the incision and expand the surgical field so as to better observe the lower limb vascular system;

[0034] (7) Using pointed forceps and fine-tipped cotton swabs, gently wipe the connective tissue to expose nerves and arteries and veins;

[0035] (8) Use a cotton swab to bluntly dissect the femoral artery, vein and nerve near the groin, being careful not to puncture the femoral vein;

[0036] (9) Insert a 7-0 suture below the proximal end of the femoral artery, with the suture as close to the proximal vessel as possible, and use double knots to block the blood flow in the proximal femoral artery;

[0037] (10) At the distal end near the knee, separate the femoral artery from the femoral vein and nerve using the same method, and ligate the distal femoral artery with 7-0 sutures;

[0038] (11) Remove the traction device, suture the muscle and skin with 4-0 sutures, and disinfect the area around the surgical incision with povidone-iodine;

[0039] (12) Carefully transfer the mice after surgery to a preheated incubator at 36°C to allow them to recover;

[0040] (13) The blood flow recovery was dynamically monitored on postoperative Day 0, Day 3, Day 5, Day 7, Day 14 and Day 21 using a blood flow meter.

[0041] Example 2: Immunohistochemical staining (gastrocnemius muscle CD31)

[0042] (1) Gastrocnemius muscle harvesting: Day 14 mice after lower limb ischemia were euthanized by cervical dislocation. The gastrocnemius muscle of the mice was completely removed with curved forceps and surgical scissors and fixed in 4% PFA solution overnight.

[0043] (2) Remove the gastrocnemius muscle tissue from the 4% PFA solution and rinse with running water to remove any remaining 4% PFA solution;

[0044] (3) Dehydration: The gastrocnemius muscle tissue was transferred sequentially to 70% ethanol, 75% ethanol, 85% ethanol, 95% ethanol (I), 95% ethanol (II), anhydrous ethanol (I), and anhydrous ethanol (II), and soaked for 30 min each time;

[0045] (4) Transparency: After dehydration, the gastrocnemius muscle tissue was transferred sequentially to xylene (I) and (II) and soaked for 30 min each to make the tissue samples transparent;

[0046] (5) Wax impregnation: Immerse the gastrocnemius muscle tissue with molten paraffin (I), (II), and (III) for 1 hour each time;

[0047] (6) Embedding: Place the wax-impregnated gastrocnemius muscle tissue into a mold, add molten paraffin wax, and place the mold on an ice platform to allow the paraffin wax to solidify;

[0048] (7) Sectioning: Ensure that the tissue embedded in paraffin has cooled and solidified sufficiently, and then section it using a microtome. The section thickness should be about 5-6 μm.

[0049] (8) Spreading: Gently scrape the tissue from the blade with a brush and spread it in 42°C warm water;

[0050] (9) Baking: Place the gastrocnemius muscle tissue slices in a 65℃ oven and bake for 6 hours;

[0051] (10) Dewaxing: Soak tissue sections in xylene (I) for 10 min, then soak them in xylene (II) for 5 min;

[0052] (11) Hydration: The tissue sections were placed in anhydrous ethanol (I), anhydrous ethanol (II), 95% ethanol (I), 95% ethanol (II), 85% ethanol and 70% ethanol in sequence, and each time was soaked once for 5 min. Finally, they were placed in ddH2O for washing and soaking for 10 min.

[0053] (12) Antigen retrieval: Dilute 50× antigen retrieval solution with PBS to 1×, place it in a microwave oven and preheat on medium-high heat until the liquid boils. Then, turn the microwave oven to medium heat. After the liquid boils again, put the slide in and microwave on medium heat for about 5-8 minutes. After the liquid boils again, turn the microwave oven to medium-low heat and wait for the liquid to boil again. Then, take out the slide and cool it to room temperature.

[0054] (13) Wash the tissue with PBS buffer three times, 5 min each time;

[0055] (14) Inactivation: Add 3% hydrogen peroxide solution to the tissue and incubate at room temperature for 10 min;

[0056] (15) Wash with PBS buffer at least 3 times, 5 min each time;

[0057] (16) Blocking: Prepare 5% BSA with PBS buffer, add it to cover the tissue area, and incubate in a humidified chamber at room temperature for 1.5 h;

[0058] (17) Primary antibody incubation: Aspirate the blocking solution, circle the primary antibody incubation area with a histochemical pen, add 10 μL of primary antibody diluted with 5% BSA to each tissue, and incubate overnight at 4°C in a humidified chamber;

[0059] (18) Wash three times with PBS buffer, 10 min each time;

[0060] (19) Secondary antibody incubation: Add diluted secondary antibody dropwise and incubate at room temperature for 60 min;

[0061] (20) Wash three times with PBS buffer, 10 min each time;

[0062] (21) Color development: Add 50 μL each of DAB buffer (20×), DAB substrate (20×) and DAB chromogen (20×) to 0.85 mL of distilled water and mix well to prepare DAB color development solution. Develop color at room temperature for 3-10 min.

[0063] (22) Counterstaining: Add 80 μL of hematoxylin staining solution to cover the tissue, incubate at room temperature for 3 min, rinse with tap water for 5-10 min to return to blue;

[0064] (23) Mounting: The sections were dehydrated for 1 min each with 70%, 85%, 95% ethanol, anhydrous ethanol (I), and anhydrous ethanol (II) in sequence, cleared twice with xylene for 1 min each, and then mounted with neutral resin.

[0065] (24) After ventilation and drying, observe under a microscope.

[0066] Example 3: RNA extraction from gastrocnemius muscle tissue and real-time quantitative PCR (qPCR)

[0067] 1) Tissue RNA extraction

[0068] (1) Weigh 50-100 mg of gastrocnemius muscle tissue into an enzyme-free grinding tube, cut it on ice, add 2-3 enzyme-free grinding beads, add 1 mL of Trizol and grind for 5 min, and then transfer the lysis buffer to a new enzyme-free centrifuge tube.

[0069] (2) Let stand at room temperature for 15 min;

[0070] (3) Add 200 μL of chloroform, shake vigorously for 30 s, and let stand in an ice bath for 10 min;

[0071] (4) Centrifuge at 12,000 rpm for 15 min at 4℃, and transfer about 500 μL of the upper aqueous phase to a new EP tube;

[0072] (5) Add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 min;

[0073] (6) Centrifuge at 4℃ and 12000 rpm for 15 min, then discard the supernatant;

[0074] (7) Wash with pre-cooled 75% ethanol, centrifuge at 7500 rpm for 5 min at 4°C, and repeat once;

[0075] (8) Discard the supernatant, air dry at room temperature until the precipitate is clear, and add 20-50 μL of DEPC water to dissolve the RNA;

[0076] (9) Determine RNA concentration and purity.

[0077] 2) Tissue RNA extraction

[0078] (1) Follow the instructions of the Vazyme reverse transcription kit: calculate the volume required for 1 μg of RNA based on the RNA concentration;

[0079] (2) Prepare a 0.2 mL enzyme-free centrifuge tube, add the volume corresponding to 1 μg RNA, 4 μL 4×g DNAwiper Mix, and then add RNase-free ddH2O to make up to 16 μL. Mix gently with a pipette and incubate in a 42°C water bath for 2 min.

[0080] (3) After the water bath, remove the centrifuge tubes and add 4 μL of 5×HisScript II qRT SuperMix II to each tube. Mix gently with a pipette.

[0081] (4) Place the centrifuge tube into the PCR instrument and set the reverse transcription reaction conditions as follows: 50 °C for 15 min, 85 °C for 5 s;

[0082] (5) After the reverse transcription reaction is completed, store the cDNA at -20°C for later use.

[0083] 3) qPCR detection

[0084] qPCR system:

[0085] reagent components volume 2×AceQ qPCRSYBR Green MasterMix 5μL Forward Primer (10μM) 0.2μL Reverse Primer (10μM) 0.2μL 50×ROX Reference Dye1 0.2μL template cDNA 2μL <![CDATA[ddH2O]]> 2.4μL

[0086] The primer sequences are as follows:

[0087]

[0088] III. Experimental Conclusions

[0089] 1. Deletion of the Ncoa3 polyQ domain significantly delays the recovery of blood perfusion after lower limb ischemia. Figure 1 Laser speckle flowmeter imaging and statistical results of the mean perfusion ratio of the lower limbs at different time points after surgery in a lower limb ischemia model showed that, compared with WT mice, Ncoa3 △Q / △Q Postoperative blood flow recovery was slow in mice, with only about 60% of the blood flow on the affected side restored by day 21 postoperatively. In contrast, blood flow in WT mice was basically restored to normal levels by day 21 postoperatively, confirming that this domain plays an important role in blood flow compensation after ischemia.

[0090] 2. Deletion of the Ncoa3polyQ domain reduces the vascular-positive area in ischemic tissue. Figure 2 Immunohistochemical images of gastrocnemius muscle tissue on postoperative day 14 showed that, compared with WT mice, Ncoa3... △Q / △Q The CD31-positive area in the gastrocnemius muscle tissue of mice was significantly reduced.

[0091] 3. Deletion of the Ncoa3polyQ domain downregulates the mRNA expression of vascular marker genes in ischemic tissue. Figure 3 qPCR results showed that, compared with WT mice, Ncoa3 △Q / △Q On day 14 post-surgery, the mRNA expression of the vascular marker gene Pecam1 in the gastrocnemius muscle tissue of mice was significantly reduced.

[0092] Based on the above experimental results, this application draws the following conclusions: The NCOA3 polyQ domain serves as an important structural basis for regulating blood perfusion recovery and collateral vessel formation after lower limb ischemia. Its functional loss can significantly delay blood perfusion recovery after ischemia, reduce CD31 positive signal in ischemic muscle tissue, and downregulate the expression of the vascular endothelial-related marker gene Pecam1. This in vivo evidence clearly establishes the key association between the NCOA3 polyQ domain and the angiogenesis process in lower limb ischemic diseases, providing experimental basis and new research directions for developing intervention strategies and related therapeutic agents targeting the NCOA3 polyQ domain (such as small molecule inhibitors, nucleic acid intervention drugs such as siRNA / antisense oligonucleotides, or gene editing intervention systems).

Claims

1. Application of NCOA3 polyQ domain as a target in the preparation of drugs to alleviate lower limb ischemic diseases.

2. The application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate lower limb ischemia according to claim 1, characterized in that: The drug is used to inhibit the function of the NCOOA3 polyQ domain or to block angiogenesis in the streets.

3. The application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate lower limb ischemia according to claim 1, characterized in that: The drug includes at least one of the following: a nucleic acid intervention agent targeting the NCOOA3 polyQ domain, a gene editing intervention system, and a small molecule compound.

4. The application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate lower limb ischemia according to claim 1, characterized in that: The lower limb ischemic diseases include peripheral artery disease-related lower limb ischemia, insufficient blood perfusion in ischemic limbs, and impaired collateral circulation formation after ischemia.

5. The application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate lower limb ischemia according to claim 1, characterized in that: The drug also includes other medically acceptable adjuvants.

6. The application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate lower limb ischemia according to claim 1, characterized in that: The drug is one of the following: injection, tablet, capsule, granule, lyophilized powder for injection, ointment, or gel.

7. A drug, characterized in that: The drug is used to relieve lower limb ischemic diseases, and the drug is used to inhibit the function of the NCOA3 polyQ domain or block angiogenesis in the street.

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