Application of BACE2 inhibitors and compositions in the preparation of drugs for arteriovenous fistula stenosis after renal failure

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the use of BACE2 inhibitors and compositions in the preparation of drugs for arteriovenous fistula stenosis after renal failure. The present invention provides the use of BACE2 inhibitors and compositions thereof in the preparation of drugs for the prevention, adjuvant treatment or treatment of arteriovenous fistula stenosis after renal failure. The drug prevents, assists in the treatment of or treats arteriovenous fistula stenosis after renal failure by reducing the expression level of BACE2, reducing the content of soluble sVEGFR3, promoting the construction of surface lymphatic network of arteriovenous fistula after renal failure, reducing inflammatory infiltration or reducing the production of new endothelium. The preparation method of the BACE2 inhibitor composition provided by the present invention is simple, can play a sustained release effect of BACE2 inhibitors, and is an effective intervention method for preventing and treating arteriovenous fistula stenosis after renal failure.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a BACE2 inhibitor and a composition in the preparation of a drug for arteriovenous fistula stenosis after renal failure. Background Art

[0002] Arteriovenous fistula (AVF) is the lifeline for hemodialysis patients with chronic renal failure, accounting for more than 80% of the vascular access of hemodialysis patients. Arteriovenous fistula stenosis and thrombosis are common complications in hemodialysis patients. Blocked arteriovenous fistulas often require multiple surgical interventions to prolong the patency time. Therefore, how to help arteriovenous fistulas safely transition to the mature stage and improve the long-term patency rate of arteriovenous fistulas is an urgent problem to be solved for hemodialysis patients.

[0003] Among the many factors that cause arteriovenous fistula stenosis, intimal hyperplasia near the anastomosis of the venous segment is the main obstacle to the maturation of the fistula. This new intima is mainly due to the continuous exposure of the venous wall to the turbulent hemodynamic environment, causing endothelial damage and aggregated inflammatory response, which then forms centripetal smooth muscle or myofibroblast proliferation or migration, and finally blocks the lumen, forming the traditional definition of proliferative stenosis (non-thrombotic stenosis). The body fluid environment of patients with renal failure (such as uremia) will aggravate the hypoxia or inflammatory response on the surface of the arteriovenous fistula, thereby promoting the occurrence of arteriovenous fistula stenosis. However, there is currently a lack of effective intervention methods to improve the long-term patency of arteriovenous fistulas in patients with renal failure.

[0004] On the other hand, as an important participant in a variety of vascular diseases, the importance of the adventitial lymphatic network in vascular inflammatory response and tissue fluid drainage has been verified by many parties. Despite this, there are still few reports on the role of the lymphatic network in arteriovenous fistula stenosis. Studies have found that the lymphatic network on the surface of the arteriovenous fistula in renal failure mice is defective, that is, it is insensitive to the key lymphangiogenesis factor VEGF-C. In response to this new discovery, corresponding treatment methods can be designed: that is, by improving the growth state of the lymphatic network on the surface of the arteriovenous fistula in the environment of renal failure, maintaining the role of new lymphatics in preventing arteriovenous fistula anastomotic stenosis. At present, there is no application of BACE2 inhibitors in the preparation of drugs for the treatment of arteriovenous fistula stenosis after renal failure. This treatment plan is expected to provide inspiration and direction for improving the utilization rate of arteriovenous fistula access and inspiring new treatment methods. Summary of the invention

[0005] In view of the above-mentioned deficiencies, the present invention provides the use of BACE2 inhibitors and compositions in the preparation of drugs for arteriovenous fistula stenosis after renal failure. The present invention provides the use of BACE2 inhibitors and compositions in the preparation of drugs for the prevention, adjuvant treatment or treatment of arteriovenous fistula stenosis after renal failure. The drug prevents, assists in the treatment of or treats arteriovenous fistula stenosis after renal failure by reducing the expression level of BACE2, reducing the content of soluble sVEGFR3, promoting the construction of the surface lymphatic network of arteriovenous fistula after renal failure, reducing inflammatory infiltration or reducing the production of new endothelium. The preparation method of the BACE2 inhibitor composition provided by the present invention is simple, and it can play a sustained release effect of the BACE2 inhibitor, which is an effective intervention method for preventing and treating arteriovenous fistula stenosis after renal failure.

[0006] The technical solution of the present invention includes:

[0007] In a first aspect, the present invention provides the use of a BACE2 inhibitor in the preparation of a medicament for preventing, assisting in the treatment or treating arteriovenous fistula stenosis after renal failure.

[0008] Specifically, the drug prevents, assists in the treatment of, or treats arteriovenous fistula stenosis after renal failure through one or more of the following mechanisms:

[0009] (1) reduce the expression level of BACE2; or

[0010] (2) reducing the amount of soluble sVEGFR3; or

[0011] (3) Promote the construction of surface lymphatic network of arteriovenous fistula after renal failure; or

[0012] (4) reduce inflammatory infiltration; or

[0013] (5) Reduce the production of new endometrium.

[0014] Preferably, the BACE2 inhibitor is BACE2-IN-1.

[0015] Preferably, the BACE2 inhibitor is the sole or main active ingredient of the drug.

[0016] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0017] Preferably, the pharmaceutically acceptable excipients include one or more of a filler, a binder, a disintegrant, a lubricant, a stabilizer, a surfactant, an antioxidant, a pH adjuster or a preservative.

[0018] Preferably, the pharmaceutically acceptable excipient comprises Pluronic F-127.

[0019] More preferably, the concentration of Pluronic F-127 is 10%-30% w / v.

[0020] Still further preferably, the concentration of Pluronic F-127 is 10%-11% w / v, 11%-12% w / v, 12%-13% w / v, 13%-14% w / v, 14%-15% w / v, 15%-16% w / v, 16%-17% w / v, 17%-18% w / v, 18%-19% w / v, 19%-20% w / v, 21%-22% w / v, 22%-23% w / v, 23%-24% w / v, 24%-25% w / v, 25%-26% w / v, 26%-27% w / v, 27%-28% w / v, 28%-29% w / v or 29%-30% w / v.

[0021] More preferably, the concentration of Pluronic F-127 is 20% w / v.

[0022] Further preferably, the ratio of Pluronic F-127 to BACE2 inhibitor is 1-3 μL:1 μg.

[0023] Still more preferably, the ratio of Pluronic F-127 to BACE2 inhibitor is 1 μL:1 μg, 2 μL:1 μg or 3 μL:1 μg.

[0024] More preferably, the ratio of Pluronic F-127 to BACE2 inhibitor is 2 μL:1 μg.

[0025] Specifically, the dosage form of the drug includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0026] Preferably, the dosage forms for administration via the gastrointestinal tract include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0027] Preferably, the non-gastrointestinal administration dosage forms include, but are not limited to, injection dosage forms, respiratory tract administration dosage forms, skin administration dosage forms, mucosal administration dosage forms and cavity administration dosage forms.

[0028] Specifically, the drug can also be used in combination with other drugs for preventing, assisting in the treatment or treating arteriovenous fistula stenosis after renal failure.

[0029] Preferably, the other drugs for preventing, assisting in the treatment or treating arteriovenous fistula stenosis after renal failure include: one or more of sirolimus, sunitinib, rapamycin, paclitaxel, folic acid, statins, calcium channel blockers, warfarin, aspirin, angiotensin converting enzyme inhibitors, clopidogrel, ticlopidine hydrochloride and dipyridamole.

[0030] Preferably, the combined use is simultaneous use or sequential use.

[0031] Preferably, the dosage of the drug is adjusted according to the severity of the disease, the age of the patient, the method of administration and the course of treatment.

[0032] In a second aspect, the present invention provides the use of a BACE2 inhibitor composition in the preparation of a medicament for preventing, assisting in the treatment or treating arteriovenous fistula stenosis after renal failure.

[0033] Specifically, the BACE2 inhibitor composition consists of a BACE2 inhibitor and Pluronic F-127.

[0034] Preferably, the concentration of Pluronic F-127 is 10%-30% w / v.

[0035] Further preferably, the concentration of Pluronic F-127 is 10%-11% w / v, 11%-12% w / v, 12%-13% w / v, 13%-14% w / v, 14%-15% w / v, 15%-16% w / v, 16%-17% w / v, 17%-18% w / v, 18%-19% w / v, 19%-20% w / v, 21%-22% w / v, 22%-23% w / v, 23%-24% w / v, 24%-25% w / v, 25%-26% w / v, 26%-27% w / v, 27%-28% w / v, 28%-29% w / v or 29%-30% w / v.

[0036] Still more preferably, the concentration of Pluronic F-127 is 20% w / v.

[0037] Preferably, the ratio of Pluronic F-127 to BACE2 inhibitor is 1-3 μL:1 μg.

[0038] Still more preferably, the ratio of Pluronic F-127 to BACE2 inhibitor is 1 μL:1 μg, 2 μL:1 μg or 3 μL:1 μg.

[0039] More preferably, the ratio of Pluronic F-127 to BACE2 inhibitor is 2 μL:1 μg.

[0040] Specifically, the preparation method of the BACE2 inhibitor composition comprises the following steps:

[0041] S1. dissolving a BACE2 inhibitor in a solvent to obtain a BACE2 inhibitor solution;

[0042] S2. Add the BACE2 inhibitor solution into Pluronic F-127 and mix well to obtain a BACE2 inhibitor composition.

[0043] Preferably, the solvent is DMSO solution.

[0044] Preferably, the BACE2 inhibitor composition is prepared at 0-30°C.

[0045] Further preferably, the BACE2 inhibitor composition is prepared at 0-10°C, 10-20°C, or 20-30°C.

[0046] Still more preferably, the BACE2 inhibitor composition is prepared at 0-10°C.

[0047] Specifically, the drug prevents, assists in the treatment of, or treats arteriovenous fistula stenosis after renal failure through one or more of the following mechanisms:

[0048] (1) reduce the expression level of BACE2; or

[0049] (2) reducing the amount of soluble sVEGFR3; or

[0050] (3) Promote the construction of surface lymphatic network of arteriovenous fistula after renal failure; or

[0051] (4) reduce inflammatory infiltration; or

[0052] (5) Reduce the production of new endometrium.

[0053] Preferably, the BACE2 inhibitor is the sole or main active ingredient of the drug.

[0054] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0055] Preferably, the pharmaceutically acceptable excipients include one or more of a filler, a binder, a disintegrant, a lubricant, a stabilizer, a surfactant, an antioxidant, a pH adjuster or a preservative.

[0056] Specifically, the dosage form of the drug includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0057] Preferably, the dosage forms for administration via the gastrointestinal tract include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0058] Preferably, the non-gastrointestinal administration dosage forms include, but are not limited to, injection dosage forms, respiratory tract administration dosage forms, skin administration dosage forms, mucosal administration dosage forms and cavity administration dosage forms.

[0059] Specifically, the drug can also be used in combination with other drugs for preventing, assisting in the treatment or treating arteriovenous fistula stenosis after renal failure.

[0060] Preferably, the other drugs for preventing, assisting in the treatment or treating arteriovenous fistula stenosis after renal failure include: one or more of sirolimus, sunitinib, rapamycin, paclitaxel, folic acid, statins, calcium channel blockers, warfarin, aspirin, angiotensin converting enzyme inhibitors, clopidogrel, ticlopidine hydrochloride and dipyridamole.

[0061] Preferably, the combined use is simultaneous use or sequential use.

[0062] Preferably, the dosage of the drug is adjusted according to the severity of the disease, the age of the patient, the method of administration and the course of treatment.

[0063] The beneficial effects of the present invention are:

[0064] The present invention provides a BACE2 inhibitor and a composition thereof for use in the preparation of a drug for preventing, assisting in the treatment of, or treating arteriovenous fistula stenosis after renal failure. The drug plays a role in preventing, assisting in the treatment of, or treating arteriovenous fistula stenosis after renal failure by reducing the expression level of BACE2, reducing the content of soluble sVEGFR3, promoting the construction of the surface lymphatic network of arteriovenous fistula after renal failure, reducing inflammatory infiltration, or reducing the production of new endothelium. The preparation method of the BACE2 inhibitor composition provided by the present invention is simple, and it can play a sustained release effect of the BACE2 inhibitor, and is an effective intervention method for preventing and treating arteriovenous fistula stenosis after renal failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The following is an analysis of the status of the lymphatic network on the surface of arteriovenous fistulas. A in the figure shows the difference in the expression levels of VEGF-C and VEGF-D on the surface of arteriovenous fistulas between the Sham (sham surgery) group and the CKD (5 / 6 nephrectomy) group. B shows the difference in the density of the lymphatic network on the surface of arteriovenous fistulas between the Sham group and the CKD group. In the figure, P value >0.05 indicates no significant difference, and P value <0.05 indicates a significant difference.

[0066] Figure 2The figure shows that CKD mouse serum inhibits the proliferation-promoting effect of VEGF-C on lymphatic endothelial cells; A in the figure shows the change in the EdU-positive ratio of HuLECs after stimulation with mouse serum; B shows the difference in absorbance levels of HuLECs after treatment with CCK-8 reagent; in the figure, P value > 0.05 represents no significant difference, and P value < 0.05 represents a significant difference.

[0067] Figure 3 Western blot analysis of HuLECs after stimulation with serum from sham and CKD mice (with or without VEGF-C) 156S ) Changes in phosphorylation levels of downstream signaling pathways; P values ​​> 0.05 in the figure represent no significant difference, and P values ​​< 0.05 represent significant difference.

[0068] Figure 4 WB and ELISA analysis of the changes in sVEGFR3 and BACE2 expression levels of HuLECs after stimulation with sham and CKD mouse serum and combined with small interfering RNA transfection (siNC negative control or siBACE2); P value > 0.05 in the figure indicates no significant difference, and P value < 0.05 indicates a significant difference.

[0069] Figure 5 WB analysis was performed to compare the differences in BACE2 or VEGF-C expression levels on the surface of arteriovenous fistula in CKD mice between the control group and the BACE2 inhibition group; P value < 0.05 in the figure indicates a significant difference.

[0070] Figure 6 BACE2 inhibitor gel alleviates the stenosis rate of arteriovenous fistula; A in the figure is a macrofluorescence staining (red VEGFR-3 positive represents lymphatic network) comparing the differences in lymphatic network density on the surface of arteriovenous fistula in CKD mice and the differences in sVEGFR3 content in serum and local arteriovenous fistula between the Vehicle control group and the BACE2 inhibition group; B is the difference in the stenosis rate of arteriovenous fistula in CKD mice between the control group and the BACE2 inhibition group; C is the difference in the proportion of SMA and CD45 positive areas in arteriovenous fistula sections in CKD mice between the control group and the BACE2 inhibition group; D is a representative immunofluorescence image; in the figure, P value > 0.05 represents no significant difference, and P value < 0.05 represents a significant difference. DETAILED DESCRIPTION

[0071] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0072] Example 1 Preparation of BACE2 inhibitor gel

[0073] 1. BACE2 inhibitor gel ingredients:

[0074] BACE2 inhibitor BACE2-IN-1 (MCE, 1676107-08-6), DMSO, 20% w / v Pluronic F-127 (PF-127, Sigma, 9003-11-6).

[0075] 2. A method for preparing a BACE2 inhibitor gel, specifically:

[0076] (1) Dissolve 50 μg of BACE2 inhibitor (BACE2-IN-1) in 10 μL of DMSO at 4°C to obtain a BACE2 inhibitor DMSO solution;

[0077] (2) Add the BACE2 inhibitor DMSO solution to 100 μL of 20% w / v Pluronic F-127 gel at 4°C and mix well to obtain the BACE2 inhibitor gel.

[0078] The preparation method of the drug gel is simple. It is liquid at 4°C and gel-like at 37°C in vivo, and can play a sustained-release role of BACE2 inhibitors.

[0079] Example 2 Establishment of mouse model of arteriovenous fistula after renal failure and analysis of the status of lymphatic network on the surface of arteriovenous fistula

[0080] 1. Male C56BL / 6 mice of about 10 weeks old were selected, and CKD (Chronic Kidney Diseases) mice were constructed by removing 2 / 3 of the left kidney and completely removing the right kidney. The sham group (Sham group) was mice of the same age, and the skin of the corresponding parts was cut but no nephrectomy was performed. About 2 weeks after the operation, the activity of mice in the CKD group decreased, they curled up, had slight tremors in their bodies, and huddled together for warmth. About 6 weeks after the operation, the activity of mice increased, their reactions were agile, and their activities were light and healthy. 8 weeks after the operation, the concentrations of creatinine and urea nitrogen (μmol / L) were detected after taking part of the serum of the mice. The significant increase of about 1-2 times was considered to be a successful construction of the CKD model.

[0081] 2. Eight weeks after surgery, mice were anesthetized by isoflurane gas. After adequate shaving and preparation of the neck skin, mice were fixed in a supine position with the neck extended. Each animal received 1 IU / g heparin at the beginning of the operation. The operation was performed under a dissecting microscope. Through a midline skin incision in the neck, the right common carotid artery and external jugular vein were dissected and exposed, the right mastoid muscle was removed, all branch vessels were ligated using 10-0 surgical sutures, and the right common carotid artery was ligated using 8-0 thread proximal to the carotid bifurcation. The external carotid artery was clamped proximally and distally, and a small phlebotomy was performed in its middle part, and the end-to-side anastomosis of the right common carotid artery and external jugular vein was performed by six interrupted sutures. Thereafter, the vascular clamp was removed and the fistula patency was verified.

[0082] 3. Four weeks after the arteriovenous fistula surgery, the arteriovenous fistula tissues of the Sham group and the CKD group were extracted and fixed overnight at 4°C. After washing with PBS three times for 1 hour each, the transplanted blood vessels were immersed in CUBIC-L solution (10% N-butyldiethanolamine and 10% Triton X-100) at 37°C for 2 days. After washing with PBS three times again, the tissues were immersed in the primary antibody (VEGFR-3, R&D, AF743, 1:50; CD31, R&D, AF3628, 1:50) solution and incubated at 4°C for 2 days. After washing with PBS three times again, the transplanted blood vessels were incubated in the secondary antibody at room temperature for 1 day and then washed with PBS three times. After that, the tissues were immersed in CUBIC-R solution (45% antipyrine + 30% nicotinamide + 0.5% n-butyldiethanolamine) for transparent treatment overnight. Images were acquired using a Leica confocal microscope, and gross images of lymphatic vessels were further 3D reconstructed and counted using Imaris 9.0.1 (Bitplane, Switzerland) software.

[0083] 4. Extract arteriovenous fistula tissue from mice in the CKD group, soak in about 100 μL of RIPA (RadioImmunoprecipitation Assay) strong lysis buffer, cut into pieces, stand at 4°C for 30 minutes, centrifuge at 12500 rpm for 10 minutes, and aspirate the supernatant of tissue lysis buffer for protein quantitative detection. According to the instructions of the relevant protein quantitative kit, use the BCA protein concentration determination kit (Beyotime) to quantify protein concentration. Subsequently, perform Western Blot (WB) detection.

[0084] Figure 1 It was demonstrated that although there was no significant difference in the expression levels of VEGF-C and VEGF-D on the surface of arteriovenous fistulas between the Sham group and the CKD group, there was an obvious defect in the lymphatic network on the surface of arteriovenous fistulas in the CKD group.

[0085] Example 2 Effect of CKD mouse serum on VEGF-C / VEGFR-3 signaling pathway

[0086] The cells used in this example are human lymphatic endothelial cells (HuLECs, provided by Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) After blood was collected from the orbits of mice in the sham group or CKD group, the blood was allowed to stand for 30 minutes and centrifuged at 1000 rpm for 10 minutes to collect the supernatant, which was the mouse serum.

[0087] Endothelial culture medium EGM-2 was used as the basic culture condition, and 5% mouse serum (Sham group or CKD group) was added as the basic group; under the above conditions, VEGF-C was added or not. 156S HuLECs were stimulated with recombinant human VEGF-C (R&D Systems, 100 ng / ml), and the proliferation of HuLECs was detected by EdU staining / CCK-8 method 2 days later, or the phosphorylation level of VEGF-C / VEGFR-3 downstream pathway was detected.

[0088] 1. EdU staining: HuLECs were cultured in 50 mM EdU solution for 2 h using the BeyoClick™ EdU Cell Proliferation Kit (Alexa Fluor 488), then fixed with 4% paraformaldehyde and stained with DAPI. EdU-labeled cells were photographed using a fluorescence microscope (Olympus), and the EdU-positive ratio was calculated.

[0089] 2. CCK-8 method (Beyotime) to detect cell proliferation: 2×10 3HuLECs cells were seeded in 96-well plates. After incubation for 6 h, non-adherent cells were removed by washing with PBS. At the indicated time points, the medium was replaced with CCK-8 solution for 2 h and then transferred to a new 96-well plate, and the absorbance was assessed at 450 nm using a SpectraMax absorbance microplate reader (Molecular Devices).

[0090] 3. Phosphorylation of ERK1 / 2, AKT, and JNK signaling pathways is the downstream pathway that HuLECs mainly activate after VEGF-C binds to VEGFR-3. It is mainly regulated by phosphorylation of the intracellular segment of VEGFR-3. The final effector molecules p-ERK1 / 2, p-AKT, and p-JNK enter the nucleus to regulate the proliferation and migration of HuLECs. Therefore, the cells of the corresponding groups were lysed in RIPA lysis buffer with the addition of protease inhibitors (Roche, 05892970001), and BCA protein quantification and WB detection were performed according to the method described in "Step 3 of Example 1". The membrane was detected by enhanced chemiluminescence (ECL) method, and the image was captured by a biospectral imaging system (UVP, Upland, CA). The ratios of p-ERK / ERK, p-AKT / AKT, and p-JNK / JNK were calculated respectively to evaluate the changes in their phosphorylation levels.

[0091] 4. Extract arteriovenous fistula tissues from mice in the CKD group, soak in 100 μL RIPA strong lysis buffer, cut into pieces, stand at 4°C for 30 min, centrifuge at 12500 rpm for 10 min, and aspirate the supernatant of tissue lysis buffer for protein quantitative detection. According to the instructions of the relevant protein quantitative kit, use the BCA protein concentration determination kit (Beyotime) to quantify protein concentration. Then perform WB detection.

[0092] Figure 2 It is proved that CKD mouse serum can effectively inhibit the proliferation effect of VEGF-C on lymphatic endothelial cells. Figure 3 It was demonstrated that CKD mouse serum can widely inhibit the downstream signaling pathways activated by VEGF-C / VEGFR-3, including the ERK / JNK / AKT phosphorylation signaling pathway.

[0093] Example 3 Effects of CKD mouse serum on sVEGFR3 and BACE2

[0094] CKD serum has a wide range of inhibition on the downstream signaling pathway of HuLECs stimulated by VEGF-C. Therefore, it can be inferred that under the stimulation of CKD serum, the normal binding of VEGF-C to VEGFR-3 on the surface of lymphatic endothelial cells is hindered by the presence of certain interfering factors. As a competitive binding molecule for free VEGF-C, soluble VEGFR3 (sVEGFR3) can effectively inhibit the normal binding of VEGF-C to VEGFR-3 receptors on lymphatic endothelial cells. The present invention extracts the total protein of HuLECs stimulated by the serum of mice in the Sham group and the CKD group, and detects the difference in sVEGFR3 expression in HuLECs stimulated by the serum of mice in the Sham group and the CKD group by incubation with a primary antibody (sVEGFR3, R&D Systems, BAF743, 1:1000; 130kDa band) and WB analysis. The sVEGFR3 concentration of HuLECs lysate was detected using an ELISA detection kit (Shanghai Yanqi Biological Co., Ltd., YQ-60647K).

[0095] sVEGFR3 is an immature extracellular peptide produced by the cleavage of lymphatic endothelial cell VEGFR-3 receptor by biological enzymes. The present invention uses a cell protein WB detection method to compare the expression difference of the classic VEGFR-3 cleavage enzyme BACE2 on HuLECs after serum stimulation in the Sham group and CKD group. At the same time, small interfering RNA (provided by Jima Gene) was transfected with Lipofectamine RNAiMAX transfection reagent (ThermoFisher, 13778150) to specifically knock down the expression of BACE2 on HuLECs, and detect whether the differential expression of sVEGFR3 in HuLECs after serum stimulation in the Sham group and CKD group depends on the increase of BACE2 (detected with the above cell protein WB method).

[0096] like Figure 4 As shown, in vitro experiments confirmed that the serum of CKD group mice can significantly upregulate the expression of soluble sVEGFR3 (a competitive inhibitory molecule for the binding of VEGF-C to VEGFR-3) and VEGFR-3 cleavage enzyme BACE2 in lymphatic endothelial cells, and knocking down BACE2 can significantly inhibit the increase in sVEGFR3 expression in lymphatic endothelial cells caused by CKD mouse serum.

[0097] Example 4 BACE2 inhibitor gel reduces BACE2 expression level in arteriovenous fistula of CKD mice

[0098] After the CKD mouse arteriovenous fistula model was completed, about 100 μL of BACE2 inhibitor gel was immediately taken and applied to the surface of the mouse arteriovenous fistula (the control group was a gel containing an equal amount of DMSO solvent), incubated for about 30 minutes, and the skin was sutured continuously with 6-0 surgical sutures after the fistula pulsation was observed to be normal. During the operation, the animals were kept warm under a heating lamp until they fully recovered, and 1.5 ml of saline solution was injected subcutaneously at the end of the operation for rehydration. Four weeks after surgery, the arteriovenous fistula tissue of the CKD group mice was extracted, infiltrated in about 100 microliters of RIPA strong lysis buffer, cut into pieces, and allowed to stand at 4°C for 30 minutes. Centrifuged at 12500 rpm for 10 minutes, and the supernatant of the tissue lysate was aspirated for protein quantitative detection and WB-related experiments. The membrane was detected by enhanced chemiluminescence (ECL) method, and the image was captured by a biospectral imaging system (UVP, Upland, CA). After being standardized by β-Actin, the effects of BACE2 inhibitor gel on the expression of VEGF-C and BACE2 in the arteriovenous fistula of CKD mice were analyzed.

[0099] Figure 5 The results showed that applying BACE2 inhibitor gel could reduce the expression level of BACE2 on the surface of arteriovenous fistula in CKD mice without changing the expression level of VEGF-C.

[0100] Example 5 BACE2 inhibitor gel alleviates arteriovenous fistula stenosis rate

[0101] The present invention further conducted a correlation analysis on the growth of the surface lymphatic network, the formation of new endothelium, and the inflammatory infiltration of the arteriovenous fistula of CKD mice in the control group and the BACE2 inhibitor gel treatment group.

[0102] 1. For the growth of lymphatic network, refer to the tissue clearing method described in "Step 3 of Example 1", use the staining combination of VEGFR-3+CD31 (primary antibody), and count the total volume of lymphatic network on the surface of arteriovenous fistula in different groups by gross imaging + three-dimensional reconstruction. At the same time, ultrasonic lysis of tissue (infiltrated in 100 microliters of saline), centrifugation and extraction of supernatant, and ELISA was used to detect the change of sVEGFR3 expression in arteriovenous fistula tissue of CKD mice after BACE2 inhibition, and the total amount of precipitated protein in the lower tissue was detected, and the ratio of sVEGFR3 expression to total protein was analyzed.

[0103] 2. To evaluate the stenosis of arteriovenous fistula, flush PBS solution through the carotid artery when collecting samples. If the blood in the external jugular vein can be seen to be washed by PBS and become a transparent lumen color or there is obvious expansion of the tube wall, it is a sign of patency. If it is still a red thrombus-like color or the tube wall collapses and is not filled, it is a sign of stenosis.

[0104] 3. For unobstructed arteriovenous fistulas, frozen pathological sections were prepared for the arteriovenous fistula samples of each group in order to analyze the neointimal area and inflammatory infiltration. The samples were fixed with 4% paraformaldehyde for 2 hours at room temperature, and then dehydrated with a sucrose solution gradient (20% / 30% / 40% mass volume ratio). After the tissues and organs settled at the bottom of the sucrose solution, they were taken out and dried, immersed in OCT for penetration for 30 minutes at room temperature, and then embedded in an embedding box containing OCT to prepare frozen sections. Cryosections were air-warmed at room temperature for about 30 minutes, permeabilized and blocked in 5% donkey serum (Solarbio®, SL050, 0.1% Triton X-100 in PBS) for 1 hour, stained with primary antibodies (SMA, Sigma, F3777, 1:500; CD45, R&D Systems, AF114, 1:50) overnight at 4°C, and then incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen, 1:500) for 1 hour, then stained with DAPI (Servicebio, G1012) and mounted in anti-quenching mounting medium (Servicebio, G1401). Isotype control primary antibodies for each host species (Invitrogen, Cat No. 31933, 02-6102, 31903, 31245) were used as negative controls, and secondary antibody controls alone were used to verify antibody specificity and eliminate background signals. Use confocal microscopy to obtain frozen sections and cell staining images, and analyze SMA using relevant software + (denoting neointima) or CD45 + (Indicating inflammatory infiltration) Area ratio, the slice area was randomly selected to avoid human error.

[0105] Figure 6 The results showed that applying BACE2 inhibitor gel can effectively restore the new lymphatic network on the surface of arteriovenous fistula in CKD mice and reduce the content of local soluble sVEGFR3 in arteriovenous fistula ( Figure 6 A in the figure); at the same time, it can effectively alleviate the stenosis rate of arteriovenous fistula and reduce CD45 + Inflammatory infiltration and SMA + Neointimal production ( Figure 6 B, C in it).

[0106] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. Use of a BACE2 inhibitor in the preparation of a drug for the prevention, adjuvant treatment or treatment of arteriovenous fistula stenosis after renal failure, characterized in that: The BACE2 inhibitor is BACE2-IN-1.

2. The use according to claim 1, characterized in that: The drug prevents, assists in the treatment or treats arteriovenous fistula stenosis after renal failure through one or more of the following mechanisms: (1) reduce the expression level of BACE2; or (2) reducing the amount of soluble sVEGFR3; or (3) Promote the construction of surface lymphatic network of arteriovenous fistula after renal failure; or (4) reduce inflammatory infiltration; or (5) Reduce the production of new endometrium.

3. The use according to claim 1, characterized in that: The BACE2 inhibitor is the sole or main active ingredient of the drug.

4. The use according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The pharmaceutically acceptable excipients include one or more of a filler, a binder, a disintegrant, a lubricant, a stabilizer, a surfactant, an antioxidant, a pH adjuster or a preservative.

6. The use according to claim 5, characterized in that: The pharmaceutically acceptable excipients include Pluronic F-127, and the concentration of Pluronic F-127 is 10%-30% w / v.

7. The use according to claim 6, characterized in that: The ratio of Pluronic F-127 to BACE2 inhibitor is 1-3 μL:1 μg.

8. The use according to claim 1, characterized in that: The dosage form of the drug includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

9. The use according to claim 8, characterized in that: The dosage forms for administration via the gastrointestinal tract include: tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils; The non-intestinal administration dosage forms include: injection dosage forms, respiratory tract administration dosage forms, skin administration dosage forms, mucosal administration dosage forms and cavity administration dosage forms.

10. The use according to claim 1, characterized in that: The drug can also be used in combination with other drugs for preventing, assisting or treating arteriovenous fistula stenosis after renal failure.

11. The use according to claim 10, characterized in that: The other drugs for preventing, assisting or treating arteriovenous fistula stenosis after renal failure include: one or more of sirolimus, sunitinib, rapamycin, paclitaxel, folic acid, statins, calcium channel blockers, warfarin, aspirin, angiotensin converting enzyme inhibitors, clopidogrel, ticlopidine hydrochloride, and dipyridamole.

12. The use according to claim 10, characterized in that: The combination is used simultaneously or successively.

13. The use according to claim 10, characterized in that: The dosage of the drug is adjusted according to the severity of the disease, the age of the patient, the method of administration and the course of treatment.

14. Use of a BACE2 inhibitor composition in the preparation of a drug for preventing, assisting in the treatment or treating arteriovenous fistula stenosis after renal failure, characterized in that: The BACE2 inhibitor is BACE2-IN-1.

15. The use according to claim 14, characterized in that: The BACE2 inhibitor composition consists of a BACE2 inhibitor and Pluronic F-127.

16. The use according to claim 15, characterized in that The concentration of Pluronic F-127 is 10%-30% w / v.

17. The use according to claim 15, characterized in that: The ratio of Pluronic F-127 to BACE2 inhibitor is 1-3 μL:1 μg.

Images