Application of transcription factor CUX1 in the preparation of products for the prevention and treatment of diabetic foot ulcers
By regulating the expression level of the transcription factor CUX1, a drug composition containing CUX1 inhibitors or expression regulators was developed, which solved the problem of unsatisfactory efficacy of existing treatments for diabetic foot ulcers, promoted ulcer healing and angiogenesis, and provided a new approach to the treatment of diabetic foot ulcers.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- AFFILIATED HOSPITAL OF NANTONG UNIV
- Filing Date
- 2026-01-28
- Publication Date
- 2026-05-26
AI Technical Summary
Existing treatments for diabetic foot ulcers are not ideal, the pathological mechanisms are complex, and there is a lack of effective treatment drugs and methods.
By using transcription factor CUX1 as a target and regulating its expression level, pharmaceutical compositions containing CUX1 inhibitors or expression regulators, including injections, topical gels, or sustained-release patches, can be developed to promote fibroblast repair.
It significantly promotes the healing of diabetic foot ulcers and enhances the regenerative capacity of wound angiogenesis, providing new targets and methods for the treatment of diabetic foot ulcers.
Smart Images
Figure CN122075693A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to the application of transcription factor CUX1 in the preparation of products for the prevention and treatment of diabetic foot ulcers. Background Technology
[0002] Diabetes mellitus is a common metabolic disease characterized by chronic hyperglycemia, which seriously endangers human health. Its main danger lies in its various complications. Data released by the International Diabetes Federation predicts that approximately 643 million adults worldwide will have diabetes by 2030. Diabetic foot ulcers are one of the serious complications of diabetes, with a global incidence rate of 6.3%. In my country, the incidence of diabetic foot ulcers is rising rapidly, showing a trend of rapid growth among low-income groups, significant gender and regional differences, and a younger age of onset.
[0003] Currently, there are many clinical treatments for diabetic foot ulcers, such as debridement, decompression, anti-infection, strict blood sugar control, and stem cell transplantation. However, due to the complex pathological mechanisms of diabetic foot ulcers, the therapeutic effects of these methods are not particularly effective or ideal. Therefore, in-depth analysis of the pathological mechanisms and the search for effective treatment drugs and methods are among the key issues that urgently need to be addressed. Summary of the Invention
[0004] The purpose of this invention is to address the problem that the pathological mechanism of diabetic foot ulcers is complex and the existing treatment methods are not particularly effective or ideal.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] Application of transcription factor CUX1 as a target in the preparation of products for the prevention and treatment of diabetic foot ulcers
[0007] Preferably, the product promotes fibroblast repair function by regulating the expression level of CUX1.
[0008] Preferably, the product is a pharmaceutical composition containing a CUX1 inhibitor or a CUX1 expression regulator.
[0009] Preferably, the dosage form of the pharmaceutical composition includes an injection, a topical gel, or a sustained-release patch.
[0010] This application also provides a verification method, which verifies the application of CUX1 in diabetic foot ulcers by verifying the expression of CUX1 in an animal model of diabetic foot ulcers and in a high-glucose and high-fat injury model.
[0011] Preferably, the method for constructing the dynamic model of diabetic foot ulcer is as follows:
[0012] (a) A rat model of T1DM was established by intraperitoneal injection of streptozotocin (STZ);
[0013] (b) Establish a lower limb ischemia model by ligating the femoral artery and femoral vein in rats;
[0014] (c) A circular ulcer with a diameter of 7 mm was created on the foot using a biopsy puncture instrument, and the size of the ulcer was photographed and recorded.
[0015] Preferably, the method for constructing the high-sugar, high-fat injury model is as follows:
[0016] (a) Fibroblasts (HFF-1) were seeded in a medium containing 25-40 mmol / L glucose and 0.2-0.4 mmol / L palmitic acid (PA);
[0017] (b) After culturing for 24-48 hours, the cell viability was verified to have decreased to 50%-70% of that in the normal group by the MTT assay.
[0018] Beneficial effects:
[0019] Existing clinical trials of candidate drugs targeting specific mechanisms of diabetic foot ulcer healing have all failed. Researchers have consistently strived to explore new pathological mechanisms and identify novel therapeutic targets. CUX1 is a transcription factor that can be regulated. This invention demonstrates significantly high expression of CUX1 in diabetic foot ulcer wound tissue. In vitro cultured fibroblasts (HFF-1) showed significantly increased CUX1 expression after high glucose and high lipid cell injury; knockdown of CUX1 expression suggests that CUX1 has a protective effect against HFF-1 injury caused by high glucose and high lipid. This invention provides a new approach for the prevention and treatment of diabetic foot ulcers and offers a new target for developing diabetic wound healing techniques. Attached Figure Description
[0020] Figure 1 This is a schematic diagram illustrating CUX1 expression in ulcer tissue of diabetic rats according to one embodiment of the present invention, wherein A. Representative photographs of wound healing in the control group and the diabetic ulcer group. (Con: normal skin tissue of T1DM rats; DFU: skin tissue of diabetic rat wounds); B. Changes in CUX1 expression in different groups detected by real-time PCR (Nor: normal rat skin tissue);
[0021] Figure 2This diagram illustrates the significant increase in CUX1 expression in HFF-1 cells after high-glucose, high-fat injury in one embodiment of the present invention. Specifically: after high-glucose, high-fat injury, real-time PCR was used to detect CUX1 gene expression in each group. B and C: HFF-1 cell proteins were extracted from different treatment groups, and CUX1 expression levels were verified by Western blot experiments and statistical analysis was performed. D: Cell viability in each group was detected using the MTT assay.
[0022] Note: Nor group, HG+PA group, blank small interfering RNA+HG+PA group, CUX1 small interfering RNA+HG+PA group.
[0023] Figure 3 In one embodiment of the present invention, transcription factor CUX1 promotes the healing of diabetic foot ulcers and angiogenesis at the wound site. A: Representative photographs of wound healing in the control group, OE-NC, and OE-CUX1 group; B: Statistical graph of wound healing rate in each group; C: CD31 immunohistochemical image and magnified image of representative wounds in each group on day 21; D: Statistical count of the number of blood vessels in CD31 immunohistochemical images in each group.
[0024] Figure 4 The diagrams illustrate how the transcription factor CUX1 enhances the tube-forming ability of HUVECs. A: Representative diagrams of HUVEC tube formation under different treatment conditions; B: Statistical diagram of the number of tube-forming nodes in HUVECs; C: Statistical diagram of tube length in HUVECs. Detailed Implementation
[0025] The present invention will be further described in detail below with reference to specific embodiments.
[0026] The application of transcription factor CUX1 as a target in the preparation of products for the prevention and treatment of diabetic foot ulcers. The products promote fibroblast repair function by regulating the expression level of CUX1. The sequence of the transcription factor CUX1 is shown in SEQ ID NO: 01.
[0027] The product is a pharmaceutical composition containing a CUX1 inhibitor or a CUX1 expression regulator, and the dosage form of the pharmaceutical composition includes an injection, a topical gel, or a sustained-release patch.
[0028] This application also provides a verification method, which verifies the application of CUX1 in diabetic foot ulcers by verifying the expression of CUX1 in an animal model of diabetic foot ulcers and in a high-glucose and high-fat injury model.
[0029] In one embodiment, the method for constructing the dynamic model of diabetic foot ulcer is as follows:
[0030] (a) A rat model of T1DM was established by intraperitoneal injection of streptozotocin (STZ);
[0031] (b) Establish a lower limb ischemia model by ligating the femoral artery and femoral vein in rats;
[0032] (c) A circular ulcer with a diameter of 7 mm was created on the foot using a biopsy puncture instrument, and the size of the ulcer was photographed and recorded.
[0033] The method for constructing the high-sugar, high-fat injury model is as follows:
[0034] (a) Fibroblasts (HFF-1) were seeded in a medium containing 25-40 mmol / L glucose and 0.2-0.4 mmol / L palmitic acid (PA);
[0035] (b) After culturing for 24-48 hours, the cell viability was verified to have decreased to 50%-70% of that in the normal group by the MTT assay.
[0036] The above content will be explained in conjunction with specific verification experiments:
[0037] I. Establishment of a rat model of ischemic foot ulceration in the lower limbs of T1DM rats
[0038] Male SD rats weighing 200±20 g were selected and acclimatized for one week before model establishment. The rats were fasted but allowed water for 10 hours. STZ-sodium citrate buffer was injected intraperitoneally at a dose of 65 mg / kg, while the control group received sodium citrate buffer intraperitoneally. Random blood glucose levels were measured by tail tip blood collection three days after model establishment. A successful model was indicated by three blood glucose levels exceeding 16.7 mmol / L and accompanied by obvious "three highs and one low" symptoms (polyuria, polydipsia, polyphagia, and weight loss). For rats with uncontrolled blood glucose, STZ was administered at a dose of 10-20 mg / kg after the animals stabilized, or STZ was injected intraperitoneally again at the standard dose (65 mg / kg) after blood glucose returned to normal. The successfully modeled T1DM rats continued to be fed for four weeks to simulate the late-stage diabetic state. Four weeks later, an ischemic foot ulcer model of the T1DM rat hind limb was established. Preoperative fasting was performed. Rats were anesthetized with 0.05 mg / kg 1% amobarbital. The rats were fixed in a supine position and disinfected. The skin was incised along the course of the right inguinal vessels, and the femoral vein and artery were carefully separated. The proximal and distal ends were ligated, cut, and the skin sutured. After disinfection with povidone-iodine, a 7 mm diameter circular incision was made in the rat's paw using a sterile biopsy puncturist, and the size of the ulcer was photographed. Different groups received 200 μL of the corresponding solution, and 1 mL of the corresponding solution was injected via the tail vein. The wound was sealed with a 3M breathable membrane and wrapped with a medical self-adhesive bandage to prevent dislodgement. Wound changes were photographed and recorded on days 0, 3, 6, 9, 12, 15, 18, and 21, and exosomes or saline were administered. The rats were observed regularly to prevent bandage dislodgement. Rats were sacrificed at 2 and 3 weeks postoperatively and stored at -80℃ or fixed in 4% paraformaldehyde.
[0039] II. Establishing a cell model of high glucose and high lipid damage:
[0040] ① The cells were divided into 10 groups based on the different concentrations of glucose and PA in the basal culture medium, as shown in Table 1.
[0041] Table 1 Grouping of cell models of high glucose and high lipid injury
[0042]
[0043] ② HFF-1 cells were grafted into 96-well plates at a density of 2×103 cells / well and HUVECs at a density of 8×103 cells / well and cultured in a cell culture incubator.
[0044] ③ After 24 hours of adhesion, discard the culture medium in the wells and wash 2-3 times with sterile PBS. Add the corresponding culture medium according to the above groups and place in a cell culture incubator for culture.
[0045] ④ After the culture is complete, remove the 96-well plate and perform the MTT assay. After 24 h, wash three times with sterile PBS, add 100 μL of complete culture medium to each well, then add 20 μL of MTT staining solution to each well, and incubate at 37°C in the dark.
[0046] ⑤ After 4 h, add 100 μL of 10% SDS solution to each well and incubate at 37°C in the dark for 12-16 h.
[0047] ⑥ Detect absorbance at 570 nm wavelength and calculate cell viability. Screen for optimal glucose and lipid concentrations and establish a cell high-glucose, high-lipid damage model.
[0048] III. MTT assay for HFF-1 cell viability:
[0049] HFF-1 at 3×10 3 MTT was seeded at a density of 5 mg / mL in 96-well plates. After 1 day of culture, different concentrations of glucose and PA medium were added. After 2 days of culture, 10 μL (5 mg / mL) of MTT was added to each well, and the plates were incubated at 37°C for 4 hours. After 4 hours, 100 μL of 20% SDS was added to each well, and the plates were incubated at 37°C for another 20 hours. The optical density (OD) value of each well was measured at 570 nm using a microplate reader. The experiment was repeated three times, with six replicates for each group. The cell viability of the normal control group was taken as 100%, and the survival percentage of cells in each experimental group was calculated. Formula = [Cell viability percentage (%) = (Cell viability of damaged group ÷ Cell viability of normal control group) × 100%].
[0050] IV. RNA extraction from wound skin tissue and cells and detection of CUX1 gene expression (real-time PCR)
[0051] Rats with successful model status were selected, anesthetized, and fixed on the operating table. The abdominal cavity was cut along the midline of the thoracic and abdominal cavities, the diaphragm was carefully cut, and the lungs were allowed to recoil before the incision was widened laterally. The ribs were then cut, and the xiphoid process was clamped with hemostatic forceps and everted to fully open the thoracic cavity and expose the heart. After the air was expelled from the perfusion needle, it was inserted into the left ventricle, and the right atrial appendage was immediately cut open. 0.9% saline was rapidly perfused until the liver and mesenteric vessels became lighter in color or even white. Wound tissue was collected and stored at -80°C. The procedure was performed according to the Invitrogen TRIZOL Reagent instructions. Real-time PCR was performed using the Takara SYBR PrimeScript RT-PCR Kit, and the procedure was performed according to the kit instructions (GAPDH was used as an internal control).
[0052] V. RNA extraction from wound skin tissue and cells and detection of CUX1 gene expression (real-time PCR)
[0053] Rats with successful model status were selected, anesthetized, and fixed on the operating table. The abdominal cavity was cut along the midline of the thoracic and abdominal cavities, the diaphragm was carefully cut, and the lungs were allowed to recoil before the incision was widened laterally. The ribs were then cut, and the xiphoid process was clamped with hemostatic forceps and everted to fully open the thoracic cavity and expose the heart. After the air was expelled from the perfusion needle, it was inserted into the left ventricle, and the right atrial appendage was immediately cut open. 0.9% saline was rapidly perfused until the liver and mesenteric vessels became lighter in color or even white. Wound tissue was collected and stored at -80°C. The procedure was performed according to the Invitrogen TRIZOL Reagent instructions. Real-time PCR was performed using the Takara SYBR Prime Script RT-PCR Kit, and the procedure was performed according to the kit instructions (GAPDH was used as an internal control).
[0054] VI. CD31 Immunohistochemical Staining
[0055] Immunohistochemistry was performed on tissue sections using rabbit anti-rat CD31 antibody to detect angiogenesis in the wound after intervention. Sections were de-OCTed, treated with 3% H2O2, and blocked with goat serum. They were then incubated overnight with primary antibody CD31 at 4°C. Next, they were incubated for 1 hour with HRP-conjugated goat anti-rabbit secondary antibody (1:200, Xavier). Antibody binding in the tissue sections was then observed by incubation with DAB substrate. After counterstaining with hematoxylin, CD31 staining of blood vessels was observed under a microscope at low magnification (5X). The three areas with the most neovascularization on each slide were selected.
[0056] Experimental results:
[0057] I. Animal Model Level: CUX1 High Expression and Impaired Healing in DFU Wound Tissue
[0058] In this application, a rat model of ischemic foot ulceration of the lower limb was successfully constructed (diabetes was induced by STZ, lower limb ischemia was constructed by ligation of femoral artery and vein, and ulceration was created by a 7mm perforator). The model rats met the symptoms of "three highs and one low" and their blood glucose was consistently ≥16.7mmol / L.
[0059] Please see Figure 1 A. The healing rate of ulcer wounds in diabetic rats (DFU group) was significantly slower than that of normal skin in T1DM rats (Con group), directly demonstrating the characteristic of impaired healing of diabetic foot ulcers; such as Figure 1 Real-time PCR results showed that the CUX1 gene expression level in the DFU group was significantly higher than that in the normal rat skin tissue (Nor group) and Con group, indicating that CUX1 is highly expressed in DFU wound tissue.
[0060] II. Cellular Model Level: CUX1 Protects Fibroblasts Damaged by High Glucose and High Lipid Intake
[0061] In this application, a high glucose and high lipid injury model of HFF-1 cells was successfully constructed (the cells were seeded in a medium containing 25-40 mmol / L glucose + 0.2-0.4 mmol / L palmitic acid, and after 24-48 hours of culture, the cell viability decreased to 50%-70% of the normal group).
[0062] Expression detection results as follows Figure 2 A (real-time-PCR) and Figure 2 As shown in B and C (Western Blot experiments and statistical analysis), the gene and protein expression levels of CUX1 in the high sugar and high fat treatment group (HG+PA group) were significantly higher than those in the normal control group (Nor group), and there was no significant difference in expression levels between the blank small interfering RNA+HG+PA group and the HG+PA group.
[0063] Please see Figure 2 D. MTT assay results showed that after knocking down CUX1 (CUX1 small interfering RNA group), the viability of HFF-1 cells damaged by high glucose and high lipid was further reduced compared with the HG+PA group and the blank small interfering RNA group, confirming that CUX1 has a protective effect on fibroblasts damaged by high glucose and high lipid.
[0064] III. CUX1 overexpression promotes DFU wound healing and angiogenesis.
[0065] Please see Figure 3 A. The CUX1 overexpression group (OE-CUX1) showed significantly faster wound shrinkage rate in diabetic foot ulcers than the control group (Con) and the negative control group (OE-NC); Figure 3 The wound healing rate statistics of B confirmed that the healing rate of the OE-CUX1 group was significantly higher than that of the control group at all time points from 0d to 21d, indicating a better healing effect.
[0066] In addition, please see Figure 3 CD31 immunohistochemical image of C (including magnified view) and Figure 3 The number of blood vessels in the DFU wound was statistically analyzed. On postoperative day 21, the number of CD31 positive blood vessels in the OE-CUX1 group was significantly higher than that in the Con group and the OE-NC group, confirming that CUX1 can promote angiogenesis in the DFU wound site.
[0067] IV. CUX1 enhances the tube-forming ability of vascular endothelial cells.
[0068] In in vitro experiments, CUX1 overexpression significantly enhanced the tube-forming ability of human umbilical vein endothelial cells (HUVECs): for example... Figure 4 As shown in Figure A, compared with the control group and the OE-NC group, the tubular structures formed by HUVECs in the OE-CUX1 group were more dense and complete; as shown in Figure A. Figure 4B's pipe node count statistics chart and Figure 4 As shown in the tube formation length statistics of C, the number of tube formation nodes and the total length of tubular structures in the OE-CUX1 group were significantly increased, revealing that CUX1 provides mechanistic support for wound angiogenesis by enhancing the tube formation ability of vascular endothelial cells.
[0069] This invention utilizes a diabetic rat model of ischemic foot ulcers in the lower limbs to demonstrate for the first time a significant increase in the expression of the transcription factor CUX1 in diabetic ulcer wound tissue. Using a high-glucose, high-lipid cell injury model, it is the first time that CUX1 expression in fibroblasts has been significantly increased after high-glucose, high-lipid injury. Furthermore, it is the first time that transcription factors have been found to have a protective effect on fibroblasts damaged by high-glucose, high-lipid injury. This invention provides a new approach for the treatment of diabetic foot ulcers and offers new targets for the development of diabetic wound healing techniques.
Claims
1. Application of transcription factor CUX1 as a target in the preparation of products for the prevention and treatment of diabetic foot ulcers.
2. The application of the transcription factor CUX1 as a target in the preparation of products for the prevention and treatment of diabetic foot ulcers according to claim 1, characterized in that: The product promotes fibroblast repair by regulating the expression level of CUX1.
3. The application of the transcription factor CUX1 as a target in the preparation of products for the prevention and treatment of diabetic foot ulcers according to claim 1, characterized in that: The product is a pharmaceutical composition containing a CUX1 inhibitor or a CUX1 expression regulator.
4. The application of the transcription factor CUX1 as a target in the preparation of products for the prevention and treatment of diabetic foot ulcers according to claim 1, characterized in that: The dosage forms of the pharmaceutical composition include injections, topical gels, or sustained-release patches.
5. A verification method, characterized in that: The verification method verifies the application of CUX1 in diabetic foot ulcers by verifying its expression in animal models of diabetic foot ulcers and high-glucose, high-fat injury models.
6. The verification method according to claim 5, characterized in that: The method for constructing the dynamic model of diabetic foot ulcer is as follows: (a) A rat model of T1DM was established by intraperitoneal injection of streptozotocin (STZ); (b) Establish a lower limb ischemia model by ligating the femoral artery and femoral vein in rats; (c) A circular ulcer with a diameter of 7 mm was created on the foot using a biopsy puncture instrument, and the size of the ulcer was photographed and recorded.
7. The verification method according to claim 5, characterized in that: The method for constructing the high-sugar, high-fat injury model is as follows: (a) Fibroblasts (HFF-1) were seeded in a medium containing 25-40 mmol / L glucose and 0.2-0.4 mmol / L palmitic acid (PA); (b) After culturing for 24-48 hours, the cell viability was verified to have decreased to 50%-70% of that in the normal group by the MTT assay.