Use of SMO inhibitor in the preparation of a drug for preventing, delaying or alleviating arteriovenous fistula access stenosis

Abstract

The present invention provides the use of SMO inhibitors in the preparation of drugs for preventing, delaying or alleviating stenosis of arteriovenous fistula accesses. The present invention creatively discovers that SMO inhibitors can protect endothelial cells under high glucose stimulation and alleviate endothelial cell dysfunction, specifically by reducing LDH, decreasing apoptosis, and reducing the production of inflammatory cytokines TNF-α, MCP-1 and IL-6. Further, the present invention discovers that SMO inhibitors can improve endothelial cell dysfunction by increasing the blood flow velocity of blood vessels, increasing the inner diameter of blood vessels, improving the change of intimal thickening of blood vessels and reducing the production of inflammatory cytokines, thereby achieving the purpose of preventing, delaying or alleviating stenosis of arteriovenous fistula accesses.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to the use of SMO inhibitors in the preparation of drugs for preventing, delaying or alleviating arteriovenous fistula access stenosis. Background Art

[0002] Diabetes mellitus (DM) is a major public health problem worldwide and an established independent risk factor for cardiovascular events and cardiovascular death. The typical feature of diabetes is hyperglycemia. The most common types of diabetes are type 1 diabetes, in which absolute insulin deficiency leads to the destruction of pancreatic cells, and type 2 diabetes, in which insulin resistance may lead to hyperglycemia. Diabetes is prone to a variety of complications that almost involve every tissue of the body, and diabetes is the main cause of high cardiovascular morbidity and mortality, blindness, renal failure, and amputation. In addition, early diagnosis of type 2 diabetes in adolescents and young people under 40 years old is associated with the severity of the disease, which can lead to the premature development of serious complications.

[0003] Diabetic nephropathy (DN) is a common complication of diabetes and the main cause of chronic kidney disease. Approximately 40% of diabetic patients will develop diabetic nephropathy, which is characterized by proteinuria, elevated blood pressure, decreased renal function, and progression to end-stage renal disease (ESRD). These sobering statistics emphasize the importance of tracing the root causes of diabetes and its complications in order to provide the best measures for intervening in the treatment of this disease. When it develops into end-stage renal disease, dialysis is required to improve the condition.

[0004] An arteriovenous fistula (AVF) is the preferred vascular access for hemodialysis in patients with end-stage renal disease. The AVF has a long service life and few complications, creating favorable conditions for hemodialysis treatment. However, the primary patency rate of AVF is relatively low. A meta-analysis showed that the primary patency rate of AVF is 60% at 1 year and 51% at 2 years. Percutaneous transluminal angioplasty (PTA) is the first-line treatment for AVF dysfunction caused by vascular stenosis. However, some patients will face secondary stenosis after the recanalization of the hemodialysis access. After PTA, restenosis caused by venous neointimal hyperplasia (VNH) leads to AVF restenosis, resulting in poor patency. Studies have shown that the secondary patency rate is 71% at 1 year and 64% at 2 years.

[0005] With the development of the economy and the extension of the average life expectancy, the prevalence of diabetes in China is increasing rapidly. Diabetes and its complications pose a threat to the health and lives of patients, even leading to disability and premature death, causing huge waste of funds and resources to society. Diabetes damages blood vessels of all sizes throughout the body. Therefore, areas where blood vessels are concentrated will become the "hard-hit areas" of diabetic complications, including the kidneys, large and medium-sized blood vessels, the retina, the nervous system, etc. The medical expenses required every year are a huge figure. The prevention and treatment of diabetes and its complications are a major public health problem faced by the present invention.

[0006] Although performing AVF for diabetic nephropathy is a treatment option and a blessing for the treatment of most patients, there are some patients who will have problems with fistula stenosis after frequent dialysis, and the patency rate is not ideal. Even after performing recanalization surgical intervention after stenosis, the secondary patency rate is not ideal. This involves the mechanism causing fistula stenosis, but the specific mechanism is not yet clear, and there is still a lack of effective preventive treatment methods and subsequent treatment targets. Therefore, the present invention urgently needs to find new treatment targets and safe and effective new drugs to relieve and reduce the key molecular events in the pathogenesis of AVF stenosis in diabetic nephropathy. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide the use of SMO (Smoothened) inhibitors in the preparation of drugs for preventing, delaying or reducing the stenosis of arteriovenous fistula access. The stenosis of the arteriovenous fistula access includes the stenosis of arteriovenous fistula in hemodialysis for diabetic nephropathy.

[0008] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides the use of SMO inhibitors in the preparation of drugs for preventing, delaying or reducing the stenosis of arteriovenous fistula access.

[0010] Preferably, the SMO inhibitor includes any one or a combination of at least two of cyclopamine, vismodegib or glasdegib. The combination of at least two, for example, the combination of cyclopamine and vismodegib, the combination of glasdegib and cyclopamine, the combination of vismodegib and glasdegib, or the combination of cyclopamine + glasdegib + vismodegib.

[0011] Preferably, the SMO inhibitor includes cyclopamine.

[0012] Preferably, the dosage form of the drug includes solution, tablet, capsule or granule.

[0013] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0014] Preferably, the adjuvant includes any one or a combination of at least two of a diluent, a disintegrant, a flavoring agent, a binder, an excipient or a filler.

[0015] The present invention also provides the use of an SMO inhibitor in the preparation of a product for preventing, delaying or alleviating arteriovenous fistula access stenosis for non-diagnostic / therapeutic purposes. The product such as an animal feed additive, etc., can be used in scientific research related to arteriovenous fistula access stenosis.

[0016] In a second aspect, the present invention provides the use of an SMO inhibitor in the preparation of a drug for preventing or treating vasculitis caused by endothelial injury.

[0017] Preferably, the SMO inhibitor includes any one or a combination of at least two of cyclopamine, vismodegib or glasdegib.

[0018] Preferably, the SMO inhibitor includes cyclopamine.

[0019] In a third aspect, the present invention provides the use of an SMO inhibitor in the preparation of a drug for maintaining or increasing the blood flow velocity in blood vessels.

[0020] The present invention also provides the use of an SMO inhibitor in the preparation of a product for maintaining or increasing the blood flow velocity in blood vessels for non-diagnostic / therapeutic purposes. The product such as an animal feed additive, etc., can be used in scientific research related to blood vessels.

[0021] Preferably, the SMO inhibitor includes any one or a combination of at least two of cyclopamine, vismodegib or glasdegib.

[0022] Preferably, the SMO inhibitor includes cyclopamine.

[0023] In a fourth aspect, the present invention provides the use of an SMO inhibitor in the preparation of a drug for increasing the inner diameter of blood vessels.

[0024] The present invention also provides the use of an SMO inhibitor in the preparation of a product for increasing the inner diameter of blood vessels for non-diagnostic / therapeutic purposes. The product such as an animal feed additive, etc., can be used in scientific research related to blood vessels.

[0025] Preferably, the SMO inhibitor includes any one or a combination of at least two of cyclopamine, vismodegib or glasdegib.

[0026] Preferably, the SMO inhibitor includes cyclopamine.

[0027] In a fifth aspect, the present invention provides the use of an SMO inhibitor in the preparation of a drug for preventing or improving vascular intimal thickening.

[0028] The present invention also provides the use of an SMO inhibitor in the preparation of a product for improving intimal thickening of blood vessels for non-diagnostic / therapeutic purposes. The product is, for example, an animal feed additive, etc., and can be used in scientific research related to blood vessels.

[0029] Preferably, the SMO inhibitor includes any one or a combination of at least two of cyclopamine, vismodegib or glasdegib.

[0030] Preferably, the SMO inhibitor includes cyclopamine.

[0031] In a sixth aspect, the present invention provides the use of an SMO inhibitor in the preparation of a drug for reducing lactate dehydrogenase.

[0032] The present invention also provides the use of an SMO inhibitor in the preparation of a product for reducing lactate dehydrogenase for non-diagnostic / therapeutic purposes. The product is, for example, an animal feed additive, etc., and can be used in scientific research related to lactate dehydrogenase.

[0033] Preferably, the SMO inhibitor includes any one or a combination of at least two of cyclopamine, vismodegib or glasdegib.

[0034] Preferably, the SMO inhibitor includes cyclopamine.

[0035] In a seventh aspect, the present invention provides the use of an SMO inhibitor in the preparation of a TNF-α antagonist, an IL-6 antagonist or an MCP-1 antagonist.

[0036] The present invention also provides the use of an SMO inhibitor in the preparation of a TNF-α antagonist, an IL-6 antagonist or an MCP-1 antagonist for non-diagnostic / therapeutic purposes. The applications are, for example, basic research related to TNF-α, IL-6 antagonists or MCP-1 antagonists, etc.

[0037] Preferably, the SMO inhibitor includes any one or a combination of at least two of cyclopamine, vismodegib or glasdegib.

[0038] Preferably, the SMO inhibitor includes cyclopamine.

[0039] In an eighth aspect, the present invention provides the use of an SMO inhibitor in the preparation of an apoptosis inhibitor for vascular endothelial cells.

[0040] The present invention also provides the use of an SMO inhibitor in the preparation of an apoptosis inhibitor for vascular endothelial cells for non-diagnostic / therapeutic purposes. The applications are, for example, basic research such as the apoptosis mechanism of vascular endothelial cells.

[0041] Preferably, the SMO inhibitor includes any one or a combination of at least two of cyclopamine, vismodegib or glasdegib.

[0042] Preferably, the SMO inhibitor includes cyclopamine.

[0043] In a ninth aspect, the present invention provides the use of an SMO inhibitor in the preparation of a food, health product or drug for lowering blood sugar.

[0044] The present invention also provides the use of an SMO inhibitor in a blood sugar-lowering product for non-diagnostic / therapeutic purposes. For example, animal feed additives, etc., can be used in scientific research related to blood sugar levels.

[0045] Preferably, the SMO inhibitor includes any one or a combination of at least two of cyclopamine, vismodegib or glasdegib.

[0046] Preferably, the SMO inhibitor includes cyclopamine.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention first studies the mechanism of high-glucose-induced vascular endothelial cell dysfunction and creatively discovers that: 1. The Hedgehog (Hh) signaling pathway in endothelial cells is highly activated under high-glucose treatment, leading to endothelial cell dysfunction; 2. High glucose induces a significant increase in the expression levels of SMO, STK36 and SHH in endothelial cells, which are key genes in the Hh signaling pathway, and among them, SMO is the gene with the highest upregulation; 3. Targeting (knocking down) the SMO gene can protect endothelial cells in a high-glucose state and improve their dysfunction (manifested as reducing LDH (lactate dehydrogenase), reducing apoptosis, increasing cell viability, promoting cell proliferation, restoring cell migration ability, and inhibiting the expression of inflammatory cytokines TNF-α, MCP-1 and IL-6).

[0049] Based on the above mechanism research, the present invention further discovers and confirms that: 1. The SMO inhibitor can protect endothelial cells induced by hyperglycemia and improve endothelial cell dysfunction (manifested as reducing LDH, reducing apoptosis, and reducing the production of inflammatory cytokines TNF-α, MCP-1 and IL-6); 2. The SMO inhibitor improves endothelial cell dysfunction by increasing the blood flow velocity of blood vessels, increasing the inner diameter of blood vessels, improving the change of intimal thickening of blood vessels and reducing the production of inflammatory cytokines, thereby achieving the purpose of preventing, delaying or alleviating the stenosis of arteriovenous fistula access; 3. Compared with other SMO inhibitors, cyclopamine (Cyc) has the best effect in protecting endothelial cells induced by hyperglycemia and improving endothelial cell dysfunction, and thus can more effectively prevent, delay or alleviate the stenosis of arteriovenous fistula access.

[0050] In the present invention, through drug (SMO inhibitor) intervention treatment in a diabetic nephropathy AVF rat model, the phenomenon of stenosis occurring in AVF was successfully alleviated, the function of vascular endothelial cells was protected, the patency rate of AVF was improved, and the service life of the dialysis access was significantly extended.

[0051] Starting from improving the stenosis of diabetic nephropathy AVF, the present invention uses an SMO inhibitor to prevent the stenosis of AVF caused by the dysfunction of vascular endothelial cells in diabetic nephropathy AVF, maintain blood flow, improve the patency rate of AVF, and significantly extend the service time of the dialysis access. Moreover, the prevention or treatment plan using the SMO inhibitor is safe, effective, and has no side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is an analysis result diagram of transcriptome sequencing of high-glucose-stimulated HUVEC (human umbilical vein endothelial cells) in Example 1; among them, NG represents normal glucose, and HG represents high glucose.

[0053] Figure 2 It is a result diagram of verifying the upregulation of the SMO gene in the Hh signaling pathway by in vitro high-glucose stimulation experiment of HUVEC in Example 1; A is the qRT-PCR analysis of SMO expression in HUVEC under different glucose concentration conditions, B is the Western blot analysis of SMO expression in HUVEC under different glucose concentration conditions. The upper diagram of B is the representative Western blot diagram, and the lower diagram of B is the quantitative evaluation of the Western blot analysis results. Man represents the osmotic pressure control group, specifically 5.5 mM glucose + 34.5 mM mannitol; 5.5 represents 5.5 mM glucose, that is, the normal glucose group; 11.1 represents 11.1 mM glucose; 25 represents 25 mM glucose; 40 represents 40 mM glucose, that is, the high-glucose group; GAPDH is the internal reference protein in the Western blot experiment.

[0054] Figure 3 It is a result diagram of knocking down the SMO gene to improve the cell viability, reduce apoptosis, and promote the proliferation of HUVEC cells under high-glucose stimulation in Example 2. A is the cell viability result; B is the cell apoptosis result; C is the fluorescence result of cell proliferation (scale bar: 50 μm), D is the quantitative result of cell proliferation; among them, shSMO represents SMO knockdown; shCtrl represents the control group, that is, without SMO knockdown; HG represents high-glucose treatment; Man is the osmotic pressure control group.

[0055] Figure 4It is the result diagram showing that SMO gene knockdown in Example 2 improves LDH release, migration ability of HUVECs induced by high glucose, and reduces the expression of its inflammatory factors; A is the result of LDH determination; B is the result of wound healing (scratch assay), the left figure represents the scratch migration diagrams at different time points (scale bar: 25 μm), and the right figure is the quantitative evaluation of the scratch migration analysis results; C is the result of qRT-PCR analysis of the expression of inflammatory factors TNF-α, MCP-1, and IL-6; where HG represents high glucose treatment; Man is the osmotic pressure control group; shSMO represents SMO knockdown; shCtrl represents the control group, that is, without SMO knockdown.

[0056] Figure 5 It is the result diagram showing the effects of different SMO inhibitors on HUVECs treated with high glucose in Example 3. A is the result of SMO expression level; B is the result of cell viability; C is the result of IL-6 expression level; D is the result of TNF-α expression level; E is the result of MCP-1 expression level; where HG represents high glucose treatment; NG represents normal glucose treatment; HG + Cyc represents intervention with Cyclopamine under high glucose treatment; HG + Vis represents intervention with Vismodegib under high glucose treatment; HG + Gla represents intervention with Glasdegib under high glucose treatment.

[0057] Figure 6 It is the result diagram showing that SMO inhibitor Cyc improves endothelial cell viability, LDH release, and cell apoptosis under high glucose stimulation in Example 3; A is the result of HUVEC cell viability; B is the result of PEC cell viability; C is the result of HUVEC LDH release; D is the result of HUVEC cell apoptosis; E is the result of PEC cell apoptosis; where PEC represents primary endothelial cells (Primary EC); HG represents high glucose treatment; NG represents normal glucose treatment; Man represents the osmotic pressure control group; HG + Cyc represents intervention with Cyclopamine under high glucose treatment.

[0058] Figure 7 It is the result diagram showing that Cyc reduces the expression of cell inflammatory factors induced by high glucose in Example 3; A, B, and C are the results of mRNA expression levels of IL-6, TNF-α, and MCP-1 in HUVECs respectively; D is the representative western blot diagram of inflammatory factors; E, F, and G are the quantitative results of western blots of IL-6, TNF-α, and MCP-1 respectively; where "-" represents without Cyc intervention, and "+" represents with Cyc intervention; 5.5 represents 5.5 mM glucose, that is, the normal glucose group; 11.1 represents 11.1 mM glucose; 25 represents 25 mM glucose; 40 represents 40 mM glucose; GAPDH is the internal reference protein in the western blot experiment.

[0059] Figure 8 It is the result graph of the SMO inhibitor Cyc in Example 3 reducing the secretion level of cellular inflammatory factors under high glucose stimulation; A, B, and C are respectively the secretion level results of IL-6, TNF-α, and MCP-1 in HUVEC; among them, "-" indicates without Cyc intervention, and "+" indicates with Cyc intervention; 5.5 represents 5.5 mM glucose, that is, the normal glucose group; 11.1 represents 11.1 mM glucose; 25 represents 25 mM glucose; 40 represents 40 mM glucose.

[0060] Figure 9 It is the result graph of the effect of Cyc on the body weight of diabetic AVF rats in Example 4.

[0061] Figure 10 It is the result graph of the effect of Cyc on the blood glucose level of diabetic AVF rats in Example 4.

[0062] Figure 11 It is the result graph of the in vitro ultrasound examination of rat AVF in Example 4. A is the blood flow ultrasound near the AVF anastomosis at 0 s, 15 s, and 30 s. B is the blood vessel diameter near the AVF anastomosis under ultrasound. C is the peak flow velocity near the AVF anastomosis under ultrasound.

[0063] Figure 12 It is the result graph of the hematoxylin-eosin staining change of the rat AVF anastomotic blood vessels in Example 4. The scale bar is 50 μm.

[0064] Figure 13 It is the immunohistochemical feature graph of SMO, IL-6, TNF-α, MCP-1, and Cleaved caspase-3 in the rat AVF tissue in Example 4. Scale bar: 50 μm.

[0065] Figure 14 It is the result graph of Cyc reducing the expression of inflammatory factors in diabetic AVF rats in Example 4; A, B, and C are respectively the levels of TNF-α, MCP-1, and IL-6 in plasma. The 0th, 1st, and 14th weeks respectively represent the normal period, the diabetic period, and the diabetic AVF period. D is the representative western blot of SMO, TNF-α, MCP-1, and IL-6 in the rat vascular AVF tissue. E, F, G, and H are respectively the quantitative evaluations of the western blot analysis results of IL-6, TNF-α, MCP-1, and SMO. Detailed implementation manners

[0066] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0067] In the following examples, unless otherwise specified, all reagents and consumables are purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and technical means used are conventional methods and means in the art.

[0068] Term Definition

[0069] The term "arteriovenous fistula (AVF)" used in the present invention refers to a small surgical procedure of vascular anastomosis, which sutures the artery near the wrist of the forearm and the adjacent vein, so that the arterial blood flows in the anastomosed vein, forming an arteriovenous fistula, which is mainly used for hemodialysis treatment, providing sufficient blood for hemodialysis treatment and guaranteeing the adequacy of dialysis treatment.

[0070] The term "endothelial cell dysfunction" used in the present invention refers to various non-adaptive changes in the function of endothelial cells, which have important effects on hemostasis, local vascular tension, redox balance, and acute and chronic inflammatory responses.

[0071] The term "diabetic nephropathy AVF rat" used in the present invention refers to an AVF model established by anastomosing the iliac artery and iliac vein after establishing a diabetes model in SD rats through surgical intervention. The construction method is shown in detail in Example 4.

[0072] The experimental methods involved in the following examples are specifically as follows:

[0073] RNA sequencing (RNA-seq)

[0074] HUVEC was inoculated in a 6-well plate (4×10 3 -1×10 5 ), and after culturing for 48 hours under different glucose treatment conditions, the cells in each treatment group were collected. The cells were washed with PBS and then TRIzol was added. RNA extraction, sequencing library construction, and sequencing were performed by Shanghai Liebing Biopharmaceutical Technology Co., Ltd. Sequencing was performed using the Illumina NovaSeq 6000 platform. The DESeq package was used to identify differentially expressed genes (DEGs). The identification criteria for differentially expressed genes were P<0.01 and FoldChange greater than 1.5.

[0075] Quantitative fluorescence PCR (qPCR)

[0076] First, total RNA was extracted from HUVEC using the Trizol method. cDNA was synthesized using the Evo M-MLV RT Premix for qPCR. Gene-specific primers (Table 1) and the Green Premix Pro Taq HS qPCRKit kit were used for quantitative fluorescence PCR. Using 2 -ΔΔCTMethod for calculating relative mRNA expression level.

[0077] Table 1 Primer sequences for quantitative real-time PCR

[0078]

[0079]

[0080] Cell apoptosis detection

[0081] HUVECs were seeded into 6-well plates and treated with different concentrations of glucose. After 48 hours of culture, the cells were collected, washed twice with cold PBS, and resuspended in labeling buffer (1×) to 1×10 6 cells / mL. 5 μL of AnnexinV-FITC and 5 μL of PI (propidium iodide) were added. The cells were mixed well and incubated in the dark at room temperature for about 15 minutes. Another 300 μL of labeling buffer was added to each tube and loaded within 1 h. Annexin-V and PI were used to label early and late apoptotic cells respectively. Annexin-V is a Ca 2+ -dependent phospholipid-binding protein with a molecular weight of 35-36 KD, which can specifically bind to phosphatidylserine with high affinity and is a sensitive indicator for detecting early apoptotic cells (early apoptotic cells are positive). PI is a nucleic acid dye that cannot penetrate intact cell membranes but can penetrate the cell membranes of cells in the middle and late stages of apoptosis and dead cells to stain the nuclei.

[0082] CCK8 assay

[0083] 1×10 4 HUVECs were seeded into each well of a 96-well plate and grouped and treated with different concentrations of sugar. 5.5 mM glucose was used as the normal sugar concentration, and Man was used as the osmotic control. After 48 hours of culture, 10 μL of CCK8 solution was added to each well and then cultured in an incubator for 0.5 - 4 hours. The experimental principle is as follows: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt is reduced by dehydrogenases in cells to a highly water-soluble yellow formazan product under the action of the electron carrier 1-methoxy-5-methylphenazinium sulfate (1-MethoxyPMS). The amount of formazan produced is proportional to the number of live cells. Finally, the absorbance at a wavelength of 450 nm was measured using a microplate reader. Within a certain range, the number of live cells is proportional to the absorbance value. In the experiment, more than 3 replicates were set for each experimental group, and the average value was taken.

[0084] Cell proliferation EdU detection

[0085] HUVECs were seeded in 96-well plates (4×10 3 -1×10 5 cells / well) and cultured for 48 hours under normal glucose (5.5 mM glucose) and high glucose (40 mM glucose) conditions, respectively. Then, according to the instructions of the cell proliferation EdU kit, EdU was used to detect newly synthesized DNA in cells, which could be double-labeled in combination with a nuclear marker (DAPI) to detect cell proliferation, and the results were observed under a fluorescence microscope.

[0086] ELISA (enzyme-linked immunosorbent assay) detection - cells

[0087] HUVECs were seeded into 6-well plates and treated with different concentrations of glucose. The supernatant was collected after 48 hours, and the expressions of inflammatory cytokines TNF-α, MCP-1, and IL-6 in the supernatant were detected by TNF-α, MCP-1, and IL-6 ELISA kits, and the operation was carried out according to the kit instructions.

[0088] LDH (lactate dehydrogenase) assay

[0089] HUVECs were seeded in 6-well plates (1.0×10 6 cells / well), treated under different glucose conditions, and after incubation for 48 hours, the supernatant was collected and the LDH activity was analyzed using an LDH cytotoxicity assay kit.

[0090] SMO gene knockdown (plasmid construction, lentivirus production, and lentivirus transduction)

[0091] A stable knockdown cell line was generated using the lentivirus system. The SMO shRNA vector was purchased from Guangzhou Yunzhou Biotechnology Co., Ltd. All constructs were confirmed by Sanger sequencing, and the shRNA targeting sequences used in this study are shown in Table 2. Lentivirus production: The lentiviral plasmid was transfected into 293T cells. Then, the lentiviral supernatant was collected and filtered through a 0.22 μM filter 48 hours after transfection. HUVECs were infected with the lentivirus, and 48 hours after infection, puromycin (2 μg / mL) was added to the medium to select positively infected cells.

[0092] Table 2 SMO shRNA targeting sequences (5'→3')

[0093]

[0094] Wound healing assay

[0095] HUVECs were seeded in 6-well plates and incubated for 48 hours under high glucose (40 mM glucose) conditions. The cells were seeded in 6-well plates and, after growing to cover the bottom of the plate, were starved in serum-free medium for 4 - 6 hours. Then, parallel scratches were made using a 100 μL sterile micropipette tip, with the tip perpendicular and not tilted. The old medium was removed, and the cells were washed with sterile PBS to remove the scratched cells, and the corresponding medium was replenished. At 0, 6, 12, and 24 hours respectively, cell migration was observed through an inverted microscope and analyzed using Image-Pro Plus 6.0 software.

[0096] Ultrasonography

[0097] The hemodynamic changes of the iliac vessels were examined using a high-frequency high-resolution small animal ultrasound imaging system with a 13 - 24Mhz linear transducer. The rats were anesthetized with chloral hydrate (0.3 mL / 100 g) and placed in the supine position. The hair in the inguinal region was removed using shaving instruments to minimize ultrasound attenuation. Under continuous anesthesia, the rats were examined by an experienced experimenter using ultrasound. The peak blood flow velocity and vessel diameter of the proximal iliac vein (venous side of the AVF) were measured using gray-scale and Doppler ultrasound. When measuring the peak blood flow velocity, the Doppler sample volume was adjusted to 0.5 mm, and the irradiation angle was kept constant at less than 60° according to the vessel diameter and direction. The vessel diameter was detected near the iliac vascular fistula anastomosis. All observed data were measured 3 times.

[0098] HE (hematoxylin-eosin) and IHC (immunohistochemistry) staining

[0099] The paraffin-embedded tissue sections were stained with HE. The vascular tissue specimens were deparaffinized and rehydrated, and antigen retrieval was performed for IHC staining. The tissue sections were blocked at room temperature for at least 1 h and then incubated overnight at 4°C with antibodies against TNF-α, MCP1, IL-6, and SMO. After careful washing, detection was performed using the DAB-HRP method and observed under a microscope. Quantification was performed according to the percentage of live cells and staining intensity.

[0100] ELISA (enzyme-linked immunosorbent assay) detection - plasma

[0101] Peripheral blood samples of rats were collected and plasma was separated. The concentrations of cytokines (TNF-α, MCP-1, and IL-6) in the plasma were measured using an ELISA kit (Jiangsu Enzyme Immuno Industry Co., Ltd., China).

[0102] Western blot (protein immunoblotting)

[0103] After the experiment, the AVF tissues of rats were collected. The tissues were enzymatically digested to isolate vascular endothelial cells. The vascular endothelial cells were lysed with a mixture of RIPA and phosphatase inhibitor, and the supernatant was obtained by centrifugation. The protein concentration was measured to prepare protein samples for electrophoresis. Transfer the membrane at 260 mA for 60 minutes. The transferred PVDF membrane was soaked in TBST buffer containing 5% skim milk powder and blocked at a rotation speed of 60 rpm for 1 hour at room temperature, and then gently washed 3 times with TBST buffer, with each wash lasting 8 - 10 minutes. Then add the corresponding primary antibody diluents (SMO, Caspase3, TNF-α, MCP-1, and IL-6), and the dilution ratio is determined according to the requirements of each antibody. Incubate overnight on a shaker at 4°C; the next day, wash 3 times with TBST buffer, with each wash lasting 8 - 10 minutes. Add the corresponding secondary antibody diluent of the anti-species source to the PVDF membrane, incubate at room temperature for 1 hour, and then gently wash 3 times with TBST buffer, with each wash lasting 8 - 10 minutes. Development: Mix the luminescent solution A and B in a 1:1 ratio, gently blot the residual moisture on the PVDF membrane with filter paper, evenly drip the mixed luminescent solution on the PVDF membrane, and analyze the protein blotting results on a gel imager.

[0104] Example 1

[0105] Mechanism of high glucose-induced vascular endothelial cell dysfunction detected at the cellular level

[0106] The present invention first studied at the cellular level the mechanism by which high glucose (HG) stimulates the production and release of cytokines and impairs endothelial cell function. Human umbilical vein endothelial cells (HUVECs) were treated with 40 mM glucose for 48 hours, and then RNA sequencing (RNA-seq) analysis was performed on the HUVECs. The results showed that: After HG treatment, extensive changes occurred in the gene expression of most metabolic genes and signaling pathways. Notably, the expression levels of SMO, STK36, and SHH, which are key genes in the Hedgehog (Hh) signaling pathway, were significantly increased by HG induction. Consistent with this result, KEGG pathway enrichment analysis also determined that the Hh signaling pathway is a highly activated pathway in HG-treated HUVECs compared with the control group (normal glucose, NG). Among the genes in the Hh signaling pathway, SMO is the gene with the highest upregulation in HG-treated HUVECs ( Figure 1 ).

[0107] To verify the above RNA-seq results, the present invention detected the mRNA expression of SMO in HUVECs treated with different conditions. Consistent with the previous results, HG treatment strongly upregulated the mRNA expression of SMO ( Figure 2 A). Western blot analysis also confirmed the upregulation of SMO at the protein level under HG stimulation ( Figure 2B). In summary, the results of Example 1 showed that: 1. The Hedgehog (Hh) signaling pathway in HUVECs was highly activated under high glucose treatment; 2. The expression levels of SMO, STK36, and SHH in HUVECs induced by high glucose were significantly increased. They are key genes in the Hh signaling pathway, and among them, SMO is the gene with the highest upregulation.

[0108] Example 2

[0109] The Hh signaling pathway leads to endothelial dysfunction

[0110] On the basis of Example 1, the present invention further studied whether the Hh signaling pathway would lead to endothelial dysfunction. First, a cell line with SMO knockdown was constructed, and their cytokine production, cell viability, proliferation, and apoptosis were examined under high glucose conditions. The results showed that under high glucose conditions, compared with the control group (the cell line without SMO knockdown), the knockout of SMO significantly increased the viability of HUVECs ( Figure 3 A), reduced the apoptosis of endothelial cells ( Figure 3 B), and promoted the proliferation of endothelial cells ( Figure 3 C). These results indicate that the knockdown of SMO can protect endothelial cells under high glucose from dysfunction.

[0111] To further determine whether knocking down SMO can restore the dysfunction of endothelial cells, the present invention detected the LDH level of HUVECs with SMO knockdown under high glucose treatment. As expected, compared with the control group, the LDH of HUVECs was significantly reduced after knocking down SMO ( Figure 4 A); it is worth noting that the migration ability of HUVECs was also significantly restored after knocking down SMO ( Figure 4 B); moreover, knocking out SMO can also significantly inhibit the expression of inflammatory cytokines TNF-α, MCP-1, and IL-6 in HUVECs ( Figure 4 C). Generally speaking, these results fully demonstrate that the Hh signaling pathway leads to endothelial cell dysfunction, and targeting SMO can protect endothelial cells in a high glucose state and improve their dysfunction.

[0112] In summary, the results of Example 2 showed that: 1. Under high glucose treatment, the Hh signaling pathway in HUVECs was highly activated, leading to endothelial cell dysfunction; 2. Targeting (knocking down) the SMO gene can protect endothelial cells in a high glucose state and improve their dysfunction (manifested as reducing LDH, decreasing cell apoptosis, increasing cell viability, promoting cell proliferation, restoring cell migration ability, and inhibiting the expression of inflammatory cytokines TNF-α, MCP-1, and IL-6).

[0113] Example 3

[0114] Screening and Functional Verification of SMO Inhibitors in the Hh Signaling Pathway

[0115] The present invention screened several SMO inhibitors, including Cyc (Cyclopamine, purchased from Guangzhou Zuoke Biotech Development Co., Ltd., MedChemExpress, HY-17024), Vis (vismodegib, purchased from Guangzhou Zuoke Biotech Development Co., Ltd., MedChemExpress, HY-10440), and Gla (glasdegib, purchased from Guangzhou Zuoke Biotech Development Co., Ltd., MedChemExpress, HY-16391). The results showed that under the condition of equal dosage (10 μM), Cyc had the best effect in inhibiting SMO expression, increasing the cell viability of HUVECs treated with high glucose, and inhibiting the expression of inflammatory factors TNF-α, MCP-1, and IL-6 in HUVECs treated with high glucose ( Figure 5 ).

[0116] To test whether the SMO inhibitor could reproduce the effect of knocking down the SMO gene, the present invention first verified the cell viability recovery of endothelial cells in the presence of Cyc (10 μM) under HG culture conditions. As expected, Cyc intervention could significantly restore the cell viability of HUVECs in the presence of HG ( Figure 6 A). Similar results were also obtained in primary endothelial cells (Primary EC, PEC) cultured under in vitro HG conditions ( Figure 6 B). In addition, the present invention also detected the expression level of LDH in HUVECs after Cyc intervention and found that Cyc intervention could reduce LDH ( Figure 6 C). Consistently, the addition of Cyc significantly reduced the apoptosis of HUVECs and cultured primary endothelial cells under high glucose conditions ( Figure 6 D-E).

[0117] Next, it was tested whether Cyc could reduce the overproduction of inflammatory cytokines. The results showed that Cyc could inhibit the expression of IL-6, MCP-1, and TNF-α at the mRNA and protein levels ( Figure 7 A-G).

[0118] In addition, ELISA (detecting the relative content of inflammatory factors in the cell culture supernatant after treating HUVECs with 10 μM Cyc) detected a decrease in the levels of TNF-α, MCP-1, and IL-6 in the Cyc group ( Figure 8A-C). In summary, these results indicate that SMO inhibitors can rescue the functions of HUVECs and primary vascular endothelial cells in vitro and reduce the production of their inflammatory cytokines. These data suggest that SMO inhibition can be used as a method to protect against hyperglycemia-induced endothelial dysfunction.

[0119] In summary, the results of Example 3 show that: 1. SMO inhibitors can protect endothelial cells induced by hyperglycemia and improve endothelial dysfunction (manifested as reduced LDH expression, decreased apoptosis, and reduced production of inflammatory cytokines TNF-α, MCP-1, and IL-6); 2. Compared with other SMO inhibitors, Cyc is the most effective in protecting endothelial cells induced by hyperglycemia and improving endothelial dysfunction.

[0120] Example 4

[0121] Treatment of Diabetic AVF Rats with Cyc

[0122] (1) Establishment of a Rat Diabetic Model

[0123] Male Sprague-Dawley rats, 6-8 weeks old and weighing 200-250 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. To induce diabetes, the rats were fasted overnight, and 65 mg / kg Streptozotocin (STZ) (USA, sigma, S0130-1G) was dissolved in citrate buffer solution (0.1 M citric acid and 0.1 M sodium citrate) and injected intraperitoneally into the rats once. The control group received an equal volume of vehicle - sodium citrate buffer (pH 4.5). Three days after STZ injection, SD rats with a fasting plasma glucose (FPG) ≥ 300 mg / dL measured by a Roche blood glucose meter (Shanghai, China, Roche, LKBIO1500) were considered diabetic and used for subsequent experiments ( Figure 10 ).

[0124] (2) Establishment of a Rat AVF Model (AVF Surgery)

[0125] After establishing the diabetes model, SD rats were used to create the AVF model. The rats were anesthetized by intraperitoneal injection of chloral hydrate (0.3 mL / 100 g) and placed on the operating table during the operation. The iliac vascular region was exposed by a 3-cm incision along the left inguinal fold and retracting the abdominal muscle tissue and other soft tissues. The operation was performed under a microscope, and the iliac artery and vein were detached from the surrounding fascia and nerves. Then, the vein was ligated distally at the exposure, and a non-traumatic clamp was applied proximally at the exposure, and cut at a 45° angle proximally to the ligation. At the site where the anastomosis was formed, a small longitudinal incision was made in the vein with a micro scalpel. The lumens of the two blood vessels were rinsed with heparinized saline, and the vein and artery were sutured end-to-side intermittently with 9-0 monofilament nylon sutures. The AVF flow was confirmed by the bright red arterial blood through the anastomosis. A weak pulse could be felt at the proximal end of the iliac vein.

[0126] (3) The rats were divided into Ctrl group, DM group, and DM + Cyc group, with 4 rats in each group, as follows:

[0127] Table 3

[0128]

[0129]

[0130] During the experiment, the body weight and blood glucose of the rats were measured weekly, peripheral blood was extracted at specific time points (weeks 0, 1, and 14) for analysis, and AVF tissues were isolated at the end of the experiment for further analysis.

[0131] (4) Effects of Cyc on body weight and blood glucose levels in diabetic AVF rats

[0132] The monitoring results of body weight showed ( Figure 9 ): The body weight of rats without diabetes (Ctrl group) gradually increased, while the body weight of diabetic rats (DM group) gradually decreased. Compared with the DM group, the body weight of diabetic rats treated with the SMO inhibitor Cyc (DM + Cyc group) remained stable over time.

[0133] The monitoring results of blood glucose levels showed ( Figure 10 ): Although the blood glucose level of diabetic rats treated with Cyc (DM + Cyc group) decreased compared with the DM group, there was still a large difference from the blood glucose level of rats without diabetes (Ctrl group), indicating that the improvement of endothelial cell dysfunction by Cyc was not achieved through the reduction of blood glucose levels.

[0134] (5) Cyc improves the peak blood flow velocity and inner diameter of blood vessels

[0135] To evaluate whether SMO inhibitors can prevent or delay fistula stenosis, the present invention performed color Doppler ultrasound examination of blood vessels on the above-mentioned groups of rats to detect changes in the arteriovenous fistula (AVF) of the vascular dialysis access ( Figure 11 A). Compared with the untreated DM group, the blood flow in the rats of the DM + Cyc group treated with Cyc was faster. The present invention also measured the blood vessel diameter at the AVF anastomosis by ultrasound. The data of the present invention show that compared with the rats without diabetes (Ctrl group), the blood vessel diameter of the untreated diabetic rats (DM group) was significantly reduced, indicating that the vascular dialysis access became narrower. Interestingly, compared with the untreated group, the blood vessel diameter at the fistula of the rats treated with Cyc (DM + Cyc group) was significantly increased, even higher than that of the rats without diabetes (Ctrl group)( Figure 11 B), and a similar trend was observed in the peak blood flow velocity( Figure 11 C), which indicates that Cyc effectively prevented the occurrence of AVF stenosis, suggesting that Cyc can prevent, delay or prevent the occurrence of AVF stenosis by improving the peak blood flow velocity and inner diameter of blood vessels.

[0136] (6) Cyc improves the change of intimal thickening of diabetic AVF blood vessels and reduces the production of inflammatory cytokines

[0137] To understand the effect of high glucose on the development of the lesion, the present invention performed hematoxylin-eosin staining on the vascular tissue at the AVF anastomosis. Compared with the rats in the non-diabetic group (Ctrl group), the intima of the rats in the diabetic group (DM group) was significantly thickened and the lumen was relatively narrow. Surprisingly, after intervention with the SMO inhibitor Cyc, the vascular intima was improved and the vascular lumen was significantly enlarged( Figure 12 ). To further study whether the effect of Cyc on stenosis prevention is related to SMO inhibition and the alleviation of endothelial cell dysfunction, the present invention performed immunohistochemical analysis on the vascular tissue around the fistula. The results showed( Figure 13 ) that compared with the rats in the non-diabetic group (Ctrl group), the expression levels of SMO, TNF-α, MCP-1, IL-6 and cleaved caspase 3 in the rats in the diabetic group (DM group) were increased, and after intervention with Cyc, the expression levels of these proteins were significantly inhibited, indicating that the intervention with Cyc reduced the production of inflammatory cytokines.

[0138] The present invention further measured the levels of inflammatory factors in the peripheral blood plasma of rats in each group by ELISA( Figure 14(A-C). Weeks 0, 1, and 14 on the graph represent the normal, diabetic, and diabetic AVF periods, respectively. As expected, the present invention found that compared with the rats in the non-diabetic group (Ctrl group), the levels of TNF-α, MCP-1, and IL-6 in the plasma of the rats in the diabetic group (DM group) were significantly increased. After Cyc intervention, these inflammatory factors were significantly reduced. In addition, the present invention performed a protein immunoblotting experiment on vascular endothelial cells isolated from the AVF tissue of rats, and also obtained the result that Cyc intervention significantly reduced the expression levels of SMO, TNF-α, MCP-1, and IL-6 ( Figure 14 (D-H). This result indicates that therapeutic inhibition of SMO can prevent the occurrence of stenosis, which may be due to the reduction of inflammatory cytokines produced by endothelial cells in hyperglycemia caused by diabetes.

[0139] In summary, the results of Example 4 show that the SMO inhibitor Cyc improves endothelial cell dysfunction by increasing the peak blood flow velocity of blood vessels, increasing the inner diameter of blood vessels, improving the change of intimal thickening, and reducing the production of inflammatory cytokines, thereby achieving the prevention, delay, or alleviation of arteriovenous fistula access stenosis.

[0140] The applicant declares that the present invention illustrates the application of the SMO inhibitor of the present invention in the preparation of a drug for preventing, delaying, or alleviating arteriovenous fistula access stenosis through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0141] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0142] In addition, it should be noted that in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. Use of an SMO inhibitor in the preparation of a drug for preventing, delaying or alleviating arteriovenous fistula access stenosis in diabetic nephropathy, characterized in that, The SMO inhibitor is cyclopamine.

2. The application according to claim 1, wherein The dosage form of the drug includes solution, tablet, capsule or granule.

3. The application according to claim 2, wherein The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that The excipients include any one or a combination of at least two of diluents, disintegrants, flavoring agents or binders.

5. Use of an SMO inhibitor in the preparation of a drug for preventing or treating vasculitis caused by endothelial damage of arteriovenous fistulas in diabetic nephropathy, characterized in that, The SMO inhibitor is cyclopamine.

6. Use of an SMO inhibitor in the preparation of a drug for maintaining or increasing the blood flow velocity in the arteriovenous fistula blood vessels of diabetic nephropathy, characterized in that, The SMO inhibitor is cyclopamine.

7. Use of an SMO inhibitor in the preparation of a drug for increasing the inner diameter of arteriovenous fistula blood vessels in diabetic nephropathy, characterized in that, The SMO inhibitor is cyclopamine.

8. Use of an SMO inhibitor in the preparation of a drug for preventing or improving intimal thickening of arteriovenous fistula blood vessels in diabetic nephropathy, characterized in that, The SMO inhibitor is cyclopamine.

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