Application of eposuline in preparation of diabetes wound healing medicine
Exisulin inhibits ferrodynamic and oxidative stress in diabetic wound cells and promotes cell proliferation and migration, solving the problem of difficult healing of diabetic wounds, and achieving rapid healing and low recurrence of wounds.
Patent Information
- Application Number
- CN202510381197.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art is difficult to effectively treat diabetic wounds, especially diabetic foot ulcers, which leads to prolonging and difficult to heal and prone to concurrent infections. Angiogenesis disorders and oxidative stress in long-term high-sugar environments have made the treatment effect poor.
Exisulin or its derivatives are used to prepare diabetic wound healing drugs by inhibiting ferrodynamic keratinocytes in high-glycemic environments, reducing oxidative stress, and promoting cell proliferation and migration.
Exishulin can reduce the expression of ferrodysfunction-related proteins and reactive oxygen levels, improve cellular function, promote the epithelialization process of diabetic wounds, improve wound healing efficiency, and reduce recurrence rate.
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Figure CN120284937A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of isixsuprine or its derivatives in the preparation of drugs for promoting diabetic wound healing, and a pharmaceutical composition containing isixsuprine or its derivatives. Background Art
[0002] Diabetic foot ulcers (DFU) are one of the most common complications in diabetic patients, and about 19 - 34% of diabetic patients will develop DFU. According to the statistics of the International Diabetes Federation, there are approximately 537 million diabetic patients globally, among which the number of Chinese patients exceeds 140 million, ranking first in the world. Under diabetic conditions, a combination of complex factors such as peripheral sensory and motor neuropathy, chronic limb ischemia, and trauma caused by persistent hyperglycemia jointly induce the occurrence and development of DFU. Currently, the clinical treatment of DFU mainly includes surgical debridement, decompression therapy, prevention and treatment of infection, and revascularization when necessary. Nevertheless, complex pathophysiological changes such as angiogenesis disorder and oxidative stress induced by the long-term hyperglycemic environment all contribute to the delayed healing of DFU wounds and the easy occurrence of infection, ultimately possibly leading to adverse outcomes such as amputation. Moreover, the 5-year mortality rate of DFU patients after amputation is as high as 50%. Even if the ulcer wounds of newly diagnosed patients heal successfully, the recurrence rate within one year can be as high as 42%. Against the background of the large base of the diabetic population in China, DFU with high prevalence, high recurrence rate, and high mortality rate is an important public health problem that cannot be ignored. Therefore, analyzing the pathophysiological mechanism of the occurrence and development process of DFU and finding possible small molecule targeted therapeutic drugs are crucial for promoting the healing of DFU wounds and reducing the disease burden.
[0003] Ferroptosis is a recently discovered form of regulated cell death (RCD), mainly caused by the production of reactive oxygen species and the imbalance of iron homeostasis, and is genetically, biochemically, and morphologically different from apoptosis, necroptosis, autophagy, and other types of RCD death. In recent years, ferroptosis has been found to be associated with diabetes and its various complications, such as type 1 diabetes, type 2 diabetes, gestational diabetes, and diabetic complications (such as diabetic nephropathy, wound healing, diabetic cardiomyopathy, diabetic retinopathy), etc. Therefore, as a new RCD death mechanism, the discovery of ferroptosis and its regulatory mechanism may contribute to the development of new therapies for various diseases. Exisulind is an atypical anti-inflammatory drug, whose mechanism of action is different from that of traditional anti-inflammatory drugs. It mainly exerts its anti-inflammatory and pro-apoptotic effects by inhibiting phosphodiesterase activity and increasing the intracellular cAMP level. In recent years, some studies have found that exisulind also has an antioxidant stress effect, which can protect cells from oxidative damage by reducing the production of reactive oxygen species. Thus, exisulind may be able to become a potential small molecule drug for the treatment of diabetic wounds. However, there is currently no literature reporting the role and specific mechanism of exisulind in the treatment of diabetic wounds, which is worthy of further exploration and research. Summary of the Invention
[0004] The present invention is made to solve the above problems, and aims to provide a new pharmaceutical use of exisulind or its derivatives, as well as a pharmaceutical composition containing exisulind or its derivatives.
[0005] In the first aspect of the present invention, there is provided the use of exisulind or its derivatives in the preparation of a drug for diabetic wound healing.
[0006] Specifically, it plays the following three aspects of mechanism roles: (1) Exisulind or its derivatives can inhibit the ferroptosis of keratinocytes in a hyperglycemic environment, such as reducing the expression levels of ferroptosis-related proteins, MDA levels, and total iron ion content, etc. (2) Reduce the oxidative stress level in the wound and cells, including the content of reactive oxygen species. As described above, the increase in reactive oxygen species is an important reaction of oxidative stress in diabetic wounds, and reducing the content of intracellular reactive oxygen species helps to promote the healing of diabetic wounds. (3) Promote cell proliferation and migration ability. The proliferation and migration functions of keratinocytes in a hyperglycemic environment are both impaired, while after treatment with exisulind, the proliferation ability of cells is enhanced, and the migration speed is also significantly accelerated.
[0007] The exisulind derivatives in the present invention refer to exisulind compounds that can exert pharmacological effects.
[0008] To verify the therapeutic effect of isixulin on diabetic wounds, the present invention verified the improvement of the function of keratinocytes and the wound-healing promotion effect on diabetic wounds by means of in vitro experiments at the cellular level and in vivo experiments at the animal level.
[0009] For the in vitro experiments on cytology, human keratinocyte line HaCat was treated with 10 μM of isixulin for 48 h. For the in vivo animal experiments, db / db diabetic mice, 8-week-old male mice, were used for the experiments. First, a full-thickness skin defect wound was constructed in the diabetic mice. The animals were randomly divided into 3 groups: a blank control group, a group treated with pure DMSO, and a group treated with the drug isixulin. DMSO or drug intervention was carried out on the 3rd day after the wound was made. Then, the wounds were photographed and sampled at different time points to observe the wound healing situation.
[0010] In the experiments on human keratinocytes HaCat, isixulin could regulate the expression levels of ferroptosis-related proteins, and reduce the levels of reactive oxygen species, malondialdehyde (MDA), and total iron ion content under the culture conditions of high glucose. At the same time, it could also improve the proliferation and migration functions of keratinocytes. Through the in vivo experiments on mice, it was found that isixulin could promote the epithelialization process of diabetic wounds and was beneficial to wound healing.
[0011] The drug for promoting the healing of diabetic wounds described in the present invention is isixulin or its derivatives as the sole active ingredient or a pharmaceutical composition containing isixulin or its derivatives.
[0012] Furthermore, the drug for promoting the healing of diabetic wounds described in the present invention can also be used in combination with other therapeutic drugs.
[0013] The drug or pharmaceutical composition described in the present invention can be made into any dosage form with pharmaceutically common excipients. For example, it can be conventional tablets, injections, capsules, etc.
[0014] The second aspect of the present invention lies in providing a pharmaceutical composition for the healing of diabetic wounds, which is composed of isixulin or its derivatives and pharmaceutically acceptable excipients.
[0015] Functions and effects of the invention
[0016] In terms of effects, the results of in vitro cell experiments showed that isixulin could regulate the expression levels of ferroptosis-related proteins, and reduce the levels of reactive oxygen species, MDA, and total iron ion content under the culture conditions of high glucose. At the same time, it could also improve the proliferation and migration functions of keratinocytes. The results of animal model experiments found that isixulin could promote the epithelialization process of diabetic wounds and was beneficial to wound healing. Therefore, the present invention provides a new basis for isixulin or its derivatives to promote the healing of diabetic wounds.
[0017] In terms of safety, isixsuprine is a non-steroidal anti-inflammatory drug with clear pharmacological effects and low side effects, so it has been relatively mature in clinical applications. However, the latest research has found that isixsuprine can also be used as a drug to promote the healing of diabetic wounds. Research shows that isixsuprine can regulate intracellular signaling pathways, inhibit ferroptosis of keratinocytes in a hyperglycemic environment, and thus promote the healing of diabetic wounds. In addition, this type of drug has also been proven to have certain antioxidant stress capabilities, and can scavenge various excessive reactive substances that may be produced during the inflammatory reaction, making isixsuprine show great potential in the clinical application of promoting the healing of diabetic wounds. Compared with other treatment methods, isixsuprine has high safety and minimizes toxic damage to other tissues, thus providing a new safe and effective option for wound healing in diabetic patients. Description of the Drawings
[0018] Figure 1 Showing that isixsuprine can regulate the levels of ferroptosis-related proteins in human keratinocytes. Among them, A, PCR detection results of key ferroptosis-related genes ACSL4 and GPX4; B, Western blot level detection of ACSL4 and GPX4 and MDA proteins.
[0019] Figure 2 Showing that isixsuprine can reduce the levels of reactive oxygen species, MDA, and total iron ion content in human keratinocytes. Among them, A, observation results under a fluorescence microscope; B, MDA level detection; C, total iron ion content detection.
[0020] Figure 3 Showing that isixsuprine can improve the proliferation and migration functions of human keratinocytes in a hyperglycemic environment. Among them, A, CCK-8 detection; B, cell scratch assay.
[0021] Figure 4 Showing that isixsuprine can promote the healing of full-thickness skin defect wounds in diabetic mice. Among them, A, wound photography; B, HE staining. Detailed Implementation Modes
[0022] The present invention will be described in detail below in conjunction with the embodiments and the drawings. However, the following embodiments should not be regarded as limiting the scope of the present invention.
[0023] Example 1: In vitro experimental study on the ferroptosis level and function of isixsuprine on human keratinocytes in a hyperglycemic environment
[0024] I. Experimental Methods
[0025] To verify the effect of exisulind on the levels of ferroptosis-related proteins in human keratinocytes under high glucose conditions, human keratinocyte HaCaT cells were first cultured in normal glucose (Normal Glucose, NG) or treated with long-term high glucose at 35 mM to establish the high glucose group (High Glucose, HG). Then, the HG group was treated with 10 μM exisulind for 48 hours.
[0026] ① Detection of key ferroptosis-related genes and expression levels:
[0027] Total RNA and total protein of keratinocytes in the NG group, HG group, and HG+Exisulind group were collected, and the expression of key ferroptosis-related proteins (ACSL4 and GPX4) was detected by PCR and Western blot techniques.
[0028] ② Reactive oxygen species (ROS) detection
[0029] HaCaT cells were seeded into 6-well plates and divided into the NG group, HG group, and HG+Exisulind group. ROS detection was performed using a ROS assay kit. A working solution containing 10 μM DCFH-DA probe was prepared with serum-free medium, and 1 ml of the working solution was added to each well and incubated in a constant temperature incubator for 40 minutes. Then, the excess working solution was washed away with PBS buffer, and fluorescence was observed using a fluorescence microscope. The fluorescence intensity was calculated using Image J software.
[0030] ③ Malondialdehyde (MDA) level detection
[0031] HaCaT cells were seeded into 6-well plates and divided into the NG group, HG group, and HG+Exisulind group. The intracellular MDA content was detected using an MDA content detection kit. Cells were digested and collected, and experiments were carried out according to the kit instructions. For every 5 million cells, 1 ml of extraction solution was added, and the cells were ultrasonically disrupted (power 200 W, ultrasound for 3 s, interval 10 s, repeated 30 times). Centrifugation was performed at 8000 g for 10 min at 4℃, and the supernatant was taken and placed on ice for further measurement. Blank tubes and assay tubes were prepared respectively, mixed well, and incubated in a 100℃ water bath for 60 min, then cooled in an ice bath and centrifuged at 10000 g for 10 min at room temperature. 200 μL of the supernatant was pipetted into a 96-well plate, and the absorbance OD values of each sample at 532 nm and 600 nm were measured using a multifunctional microplate reader. The MDA content was calculated according to the formula in the instructions.
[0032] ④ Total iron ion content detection
[0033] HaCaT cells were seeded into 6-well plates and divided into an NG group, an HG group, and an HG+Exisulind group. A ferric ion assay kit was used to detect the total ferric ion content in the cells. The cells were digested and collected, and the experiment was carried out according to the kit instructions. For every 10 million cells, 1 mL of normal saline was added, and the cells were sonicated (power 200 W, sonication for 3 s, interval 5 s, repeated 20 times). After centrifugation at 3500 r for 10 minutes, the supernatant was taken for measurement. Blank tubes, standard tubes, and assay tubes were prepared respectively, mixed well, and then placed in a boiling water bath for 5 minutes, cooled with running water, and centrifuged at 3500 r for 10 minutes. 100 μL of the supernatant was pipetted into a 96-well plate, and a multifunctional microplate reader was used to measure the absorbance OD value of each sample at 520 nm. The total ferric ion content was calculated according to the formula in the instructions.
[0034] ⑤ Detection of cell proliferation ability
[0035] HaCaT cells were seeded into 96-well plates at a density of 5×10 3 cells / well and divided into an NG group, an HG group, and an HG+Exisulind group. A Cell Counting Kit-8 was used to measure the cell proliferation activity. At different time points, 100 μL of medium containing 10% CCK-8 reagent was added to each well and incubated in a constant temperature incubator for 2 hours. The absorbance OD value at 450 nm was detected using a multifunctional microplate reader.
[0036] ⑥ Detection of cell migration ability
[0037] HaCaT cells were seeded into 6-well plates and divided into an NG group, an HG group, and an HG+Exisulind group. They were cultured under corresponding conditions according to the experimental design. When the cell confluence reached 90%, a scratch was made perpendicular to the bottom of the plate with the tip of a pipette tip, and then cultured in the corresponding complete medium with low serum to inhibit cell proliferation. Photographs were taken at different time points through an inverted microscope. The wound healing rate was measured using Image J software, and the formula was: Wound healing rate = (Initial wound area - Wound area at different time points) / Initial wound area × 100%.
[0038] II. Experimental results
[0039] As Figure 1 shown, in a high-glucose environment, the mRNA and protein levels of the ferroptosis-related protein ACSL4 were significantly increased, while the mRNA and protein levels of GPX4 were significantly decreased. After treatment with Exisulind, the mRNA and protein levels of ACSL4 were significantly decreased, while the mRNA and protein levels of GPX4 were significantly increased. As Figure 2As shown, after high-glucose treatment, the levels of reactive oxygen species, MDA, and total iron ions in keratinocytes increased significantly. After Exisulind treatment, the level of reactive oxygen species decreased, and the levels of MDA and total iron ions also decreased significantly. As Figure 3 shown, after high-glucose treatment, the proliferation and migration functions of keratinocytes were impaired. After Exisulind treatment, the proliferation ability of the cells increased, and the migration speed also increased significantly.
[0040] Example 2: In vivo experimental study on the promotion of full-thickness skin defect wound healing in diabetic mice by Exisulind.
[0041] I. Experimental method:
[0042] Eight-week-old male db / db mice were selected for the experiment. The mice were divided into a blank control group, a simple DMSO treatment group, and a drug Exisulind treatment group. On DAY 0, full-thickness skin defect wounds with a diameter of 8 mm were constructed on both sides of the spinal line on the back of the mice. On DAY 3, DMSO or Exisulind was injected around the wounds. Then, the wound conditions were observed every other day and photographed. On DAY 7 and DAY 9 of the wounds, samples were taken for HE staining to clarify the epithelial crawling on the wounds.
[0043] II. Experimental results
[0044] As Figure 4 shown, by macroscopic observation of the wounds, it can be seen that compared with the blank control group and the simple DMSO treatment group, the wound healing in the Exisulind treatment group was accelerated. At the same time, HE staining could clarify that the epithelial crawling in the Exisulind treatment group was significantly increased.
[0045] In the above example, Exisulind was taken as an example to explore the effects of Exisulind on the ferroptosis level and function of keratinocytes, and at the same time, its promoting effect on the wounds of diabetic mice was studied. However, the present invention is not limited to Exisulind, and Exisulind derivatives that can play the same pharmacological role also belong to the protection scope of the present invention.
[0046] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.
Claims
1. Use of ebselen or its derivatives in the preparation of drugs for diabetic wound healing.
2. The use according to claim 1, characterized in that: Among them, The drug for diabetic wound healing is a drug that inhibits ferroptosis and oxidative stress levels of keratinocytes in a hyperglycemic environment, or promotes cell proliferation and migration ability.
3. The use according to claim 2, characterized in that: Among them, The drug that inhibits ferroptosis of keratinocytes in a hyperglycemic environment is a drug that reduces the expression levels of ferroptosis-related proteins, MDA levels and total iron ion content.
4. The use according to claim 2, characterized in that: Among them, The drug that inhibits oxidative stress levels is a drug that reduces the content of intracellular reactive oxygen species.
5. The use according to any one of claims 1 to 4, characterized in that: Among them, The drug for diabetic wound healing is a pharmaceutical composition containing ebselen or its derivatives.
6. The use according to claim 5, characterized in that: Among them, The drug for diabetic wound healing uses ebselen or its derivatives as the sole active ingredient.
7. The use according to claim 1, characterized in that: Among them, The drug for diabetic wound healing is a tablet, capsule or injection.
8. The use according to claim 1, characterized in that: Among them, The drug for diabetic wound healing is used in combination with other drugs that promote diabetic wound healing.
9. A pharmaceutical composition for promoting diabetic wound healing, characterized in that, It is composed of ebselen or its derivatives and pharmaceutically acceptable excipients.