Use of a trichosanthin derivative in the preparation of a medicament for treating diabetic wounds

Abstract

The application discloses application of a spinfloss lactone in preparation of a medicine for treating a wound of diabetes, and proposes that the spinfloss lactone can stimulate an iron death inhibiting protein (FSP1 protein) to inhibit iron death, and based on a treatment evaluation method of a model system, it is verified that the spinfloss lactone has good efficacy of promoting wound healing of a diabetic mouse.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the application of a traditional Chinese medicine monomer, Inula lactone, in the preparation of a drug for treating diabetic wounds. Background Technology

[0002] Diabetes has become a global public health challenge, with its prevalence continuing to rise. It also causes a variety of serious complications, particularly diabetic foot ulcers (DFUs). Approximately 34% of patients with type 1 or type 2 diabetes develop this complication during disease progression, about 50%–60% of ulcers become infected, and about 20% of moderate to severe infections lead to lower limb amputation. Globally, approximately 18.6 million patients are affected by this disabling complication each year. Currently, surgical debridement, antibiotic treatment, reducing weight-bearing pressure on the ulcer, and treating lower limb ischemia and foot infections are the first-line treatments for diabetic foot ulcers.

[0003] However, while debridement removes necrotic tissue, it may also damage healthy tissue that is still alive, which may affect healing. Diabetic foot ulcers are prone to infection, and long-term use of antibiotics can lead to bacterial resistance, making treatment more difficult. Weight-bearing reduction methods have strict requirements on patients' daily activities, causing great inconvenience to their lives, and patients often find it difficult to adhere to them in the long term. The above treatment methods may have obvious drawbacks or be difficult to implement.

[0004] Inula lactones are natural sesquiterpene lactones isolated from Inula japonica and can be used to relieve cough and sputum. Recent studies have shown that Inula lactones possess excellent anti-inflammatory, antioxidant, and antitumor activities. In triple-negative breast cancer, Inula lactones can inhibit the proliferation, migration, and invasion of MDA-MB-231 and SUM-159 cells by inhibiting the nuclear factor-jB (NF-κB) pathway. It can also exert its antitumor effect in gastric cancer through an NF-κB-mediated immune response. Furthermore, Inula lactones have been identified as an effective natural immunosuppressant targeting NLRP3, inhibiting the activation of the NLRP3 inflammasome in an NF-κB-independent manner to exert an anti-inflammatory effect. Based on the anti-inflammatory and antioxidant effects of Inula lactones, it is predicted that they may have a protective effect on diabetic wounds and promote wound healing; however, no research has yet been found on the application of Inula lactones in the treatment of diabetic foot ulcers.

[0005] In summary, the difficulty in healing diabetic foot ulcers is mainly due to oxidative stress and ferroptosis in cells. Current conventional treatments cannot effectively intervene in this mechanism. Therefore, there is an urgent need to develop a drug for the treatment of diabetic foot ulcers that can effectively inhibit ferroptosis, improve oxidative stress, and enhance the quality of wound healing in patients. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide an application of Inula lactone in the preparation of a drug for treating diabetic wounds, so as to promote the healing of diabetic wounds and solve the problems of high recurrence rate and high risk of amputation of diabetic foot ulcers.

[0007] Therefore, the present invention is implemented using the following technical solution: The use of a type of inula lactone in the preparation of a drug for treating diabetic wounds, characterized in that it is used in a drug for promoting the healing of diabetic wounds.

[0008] Furthermore, the inula lactone is a natural sesquiterpene lactone compound isolated from Inula japonica, and the chemical substance registration number of the inula lactone is No. 33627-28-0.

[0009] Furthermore, the inula lactone is the only active ingredient in the drug 3, and the drug is in the form of a solution, which is prepared by dissolving the inula lactone in 10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 300 (PEG300) and 50% physiological saline.

[0010] Furthermore, in a model-based treatment evaluation method, the inula lactone inhibits ferroptosis by stimulating the ferroptosis inhibitor protein (FSP1 protein).

[0011] Furthermore, the inula lactone promotes the repair of diabetic wounds by resisting oxidative stress and inhibiting ferroptosis.

[0012] A treatment evaluation method for the application of the aforementioned Inula lactone in the preparation of a drug for treating diabetic wounds, characterized in that: the treatment evaluation method of the model system includes the following steps: constructing a model system, adding Inula lactone to the model system for treatment, so as to systematically evaluate the effect of Inula lactone in treating diabetic wounds.

[0013] Furthermore, the model system includes a type 2 diabetic mouse model and a normal mouse model.

[0014] Furthermore, based on a type II diabetic mouse model, the dosage of the inula lactone was 10-20 mg / kg / day.

[0015] Furthermore, high glucose combined with palmitic acid (PA) was added to the model system to simulate the wound environment of type 2 diabetes. The oxidative stress of each group was assessed using dichlorofluorescein-DA dye under a fluorescence microscope and flow cytometry. DCFH-DA dye is oxidized to green fluorescent substances by reactive oxygen species. The intensity of the green fluorescence was analyzed to determine the antioxidant capacity of Inula lactone. The lipid peroxidation fluorescent probe (C11(581 / 591)) was used to assess the lipid oxidative stress of each group under a fluorescence microscope and flow cytometry. C11(581 / 591) changes color with the degree of lipid peroxidation. By analyzing its color change, it was determined whether Inula lactone could prevent oxidative damage and effectively inhibit ferroptosis. The iron overload of each group was assessed using a ferrous ion fluorescent probe (ferro orange) dye under a fluorescence microscope. Ferro orange dye produces red fluorescence after binding with iron ions to determine the intracellular iron ion concentration, thus evaluating the role of Inula lactone in anti-oxidative stress and anti-ferroptosis.

[0016] The beneficial effects of adopting the above technical solution are as follows: 1. This invention systematically evaluates the promoting effect of Inula lactone on the healing of diabetic wounds through a multi-model system. It finds that Inula lactone can inhibit ferroptosis by activating ferroptosis inhibitor protein (FSP1 protein), thereby promoting wound repair and healing.

[0017] This invention provides a safe and controllable drug component and application scheme for the healing of diabetic wounds. Inula lactone is a natural active compound with a clear source, clear structure, and low potential toxicity, and has good promotional value. Attached Figure Description

[0018] The present invention includes the following figures: Figure 1 The graph shows the effect of different concentrations of Inula lactone provided in Example 1 on wound healing in type 2 diabetic mice at different time points. Figure 2 Line graphs showing the wound healing rates of different concentrations of Inula lactone provided in Example 1 at different time points in type 2 diabetic mice. Figure 3 Bar chart showing the wound healing rate of different concentrations of Inula lactone provided in Example 1 at different time points in type 2 diabetic mice; Figure 4 The effects of Inula lactone provided in Example 2 on antioxidant stress at the cellular level in different groups; Figure 5 The effects of Inula lactone provided in Example 2 on anti-lipid peroxidation at the cellular level in different groups; Figure 6The effect of Inula lactone provided in Example 2 on iron overload resistance at the cellular level in different groups. Detailed Implementation

[0019] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0020] Inula lactone is a natural sesquiterpene lactone compound isolated from Inula japonica, possessing anti-inflammatory and antioxidant properties. This invention provides an application and evaluation method for Inula lactone in the preparation of a treatment for diabetic wounds. A model-based treatment evaluation method was employed, establishing a type 2 diabetic mouse model. Different concentrations of Inula lactone were prepared and injected subcutaneously into different groups of mouse models. Finally, based on the experimental results, the effect and efficacy of Inula lactone in the repair of diabetic wounds were systematically evaluated.

[0021] In the preliminary experiments of this invention, the concentration and dosage of Inula lactone were tested. The experiments showed that when the local administration dose exceeded 25 mg / kg, there were symptoms such as non-absorption and ulceration in the injection area. Therefore, the administration concentration should be less than 2.5%.

[0022] Animal grouping and model establishment: Eight-week-old male C57BL / 6 mice weighing 18-20 grams were used. After one week of acclimatization, the mice were randomly divided into the following groups: (1) blank group (2) untreated type II diabetic mice (T2DM group), (3) low-dose group (10 mg / Kg) treated with inula lactone (10 mg / Kg) type II diabetic mice (T2DM+Bri10 mg / Kg group) (4) high-dose group (20 mg / Kg) treated with inula lactone (20 mg / Kg) type II diabetic mice (T2DM+Bri20 mg / Kg group), with at least 6 mice in each group. The type II diabetic mouse and wound model were established as follows: After feeding the mice with a high-fat diet for 6 weeks, the mice were fasted for 12-16 hours and then injected intraperitoneally with streptozotocin (STZ, 100 mg / kg) for 3 consecutive days. Mice were fasted for 4–6 hours after injection, and their blood glucose levels were measured. Mice with blood glucose levels higher than 11.6 mmol / L were identified as diabetic models.

[0023] Drug preparation: Dissolve Inula lactone in 10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 300 (PEG300), and 50% physiological saline to prepare a solution. DMSO is soluble in water and most organic solvents such as ethanol, acetone, and benzene; PEG300 can be used to dissolve water-insoluble active pharmaceutical ingredients, and is applicable to injections, oral solutions, etc.

[0024] Storage of pharmaceutical preparations: Inula lactone powder should be stored at 4 degrees Celsius away from light. It is best to use the prepared drug solution on the same day. If storage is required, it should be placed in an environment of -20 degrees Celsius and protected from light.

[0025] Example 1: Type II diabetic mice were constructed, and the wound healing process was observed by administering the drug to systematically evaluate the effect of Inula lactone on promoting the repair of diabetic wounds.

[0026] 1. Administration Method: After successful model establishment, a one-week stabilization period was given, followed by full-thickness skin excision wounds on all diabetic mice. Mice were anesthetized by intraperitoneal injection of 1.25% tribromoethanol (0.2 ml / 10 g) combined with isoflurane inhalation anesthesia. Before the procedure, hair on the back was removed with depilatory cream, and after routine skin disinfection, a single circular full-thickness defect (10 mm in diameter) was prepared in the midline of the back using a skin punch. Subsequently, according to the grouping protocol, Inula lactone was injected subcutaneously daily for 12 days (the main drug solvent for the blank group and the II diabetic group). During the experiment, the general condition of the mice was observed daily, and the wound healing process was recorded by photography.

[0027] 2. Assessment Methods: Wound images were taken on days 0, 3, 7, 9, and 12 after wound formation in each experimental group. Wound area was quantified using ImageJ software, and the wound healing rate was calculated using the following formula for inter-group comparison: Healing rate (%) = [(Initial wound area - Unhealed wound area) / Initial wound area] × 100. Wound tissue was harvested on day 12 for embedding and sectioning, and wound healing was assessed at the histopathological level. Immunohistochemical staining was used to evaluate relevant tissue indicators, such as changes promoting fibrosis and angiogenesis.

[0028] 3. Experimental Results: Table 1. Effects of Inula lactone on wound healing rate at different time points in the control group. Table 2. Effects of Inula lactone on wound healing rate at different time points in the T2DM group. Table 3. Effects of Inula lactone on wound healing rate at different time points in the (T2DM+Bri 10mg / Kg) group. Table 4. Effects of Inula lactone on wound healing rate at different time points in the (T2DM+Bri 20mg / Kg) group. Five valid samples were taken from each group, resulting in Tables 1-4 and 5. Figure 1-3As can be seen from the chart, the wound healing rate in the treatment group was significantly higher than that in the untreated diabetic model group. Furthermore, the wound healing rate in the treatment group injected with Inula lactone was significantly improved on the seventh and twelfth days. Inula lactone significantly promoted wound healing in type 2 diabetic mice. However, there was no significant difference between high and low doses of Inula lactone.

[0029] Example 2: Constructing type II diabetic mice to observe and evaluate the effects of Inula lactone on oxidative stress and anti-ferroptosis. 1. Evaluation method: High glucose combined with palmitic acid (PA) stimulation was used to simulate the wound environment of type 2 diabetes. Mouse fibroblasts were used to simulate wound cells. The cells were divided into (1) blank group (2) high glucose + PA stimulation group (3) high glucose + PA + Bri (2.5μM) (4) High glucose + PA + Bri (5μM) was used to assess oxidative stress in each group using dichlorofluorescein (DCFH-DA) dye under fluorescence microscopy and flow cytometry. DCFH-DA dye is oxidized to green fluorescent substances upon encountering reactive oxygen species (ROS). Analyzing the green fluorescence intensity of each experimental group can determine whether the antioxidant capacity of Inula lactone can effectively scavenge excess ROS to alleviate the effects of high glucose on cells. A lipid peroxidation fluorescent probe (C11 (581 / 591)) was used to assess lipid oxidative stress in each group under fluorescence microscopy and flow cytometry. C11 (581 / 591) can detect the level of lipid peroxidation in living cells and changes color with the degree of lipid peroxidation. Analyzing its color change can determine whether Inula lactone can prevent oxidative damage and effectively inhibit ferroptosis. A ferrous ion fluorescent probe (ferro orange) was used to assess iron overload in each group under fluorescence microscopy. Ferro orange dye produces red fluorescence after binding with iron ions, which can be used to determine the intracellular iron ion concentration.

[0030] 2. Experimental Results: The results obtained from the experiment... Figure 4-6 ,from Figure 4 In studies, it was observed that inula lactone could improve oxidative stress caused by high glucose levels. Figure 5 It was observed that Inula lactones can exert anti-lipid peroxidation effects, such as... Figure 6 Intradermal lactone was observed to improve iron overload.

[0031] In conclusion, appropriate concentrations and dosages of Inula lactone have a good promoting effect on the healing of diabetic wounds. The experimental results show that diabetic wounds exhibit significant oxidative stress due to the high-glucose environment, and ferroptosis plays a crucial role in the slow healing of diabetic wounds. Inula lactone has anti-oxidative stress effects, can inhibit ferroptosis, and has a positive effect on the healing of diabetic wounds.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of a type of inula lactone in the preparation of a drug for treating diabetic wounds, characterized in that: it is Application in drugs that promote the healing of diabetic wounds.

2. The application of the inula lactone according to claim 1 in the preparation of a drug for treating diabetic wounds, characterized in that: The Inula lactone is a natural sesquiterpene lactone compound isolated from Inula japonica, and its chemical registration number is No. 33627-28-0.

3. The application of the inula lactone according to claim 2 in the preparation of a drug for treating diabetic wounds, characterized in that: The inula lactone is the only active ingredient in the drug, which is a solution prepared by dissolving the inula lactone in 10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 300 (PEG300), and 50% physiological saline.

4. The application of the inula lactone according to claim 2 or 3 in the preparation of a drug for treating diabetic wounds, characterized in that: The treatment evaluation method based on the model system states that the inula lactone inhibits ferroptosis by stimulating the ferroptosis inhibitor protein (FSP1 protein).

5. The application of the Inula lactone according to claim 4 in the preparation of a drug for treating diabetic wounds, characterized in that: The inula lactone promotes the repair of diabetic wounds by resisting oxidative stress and inhibiting ferroptosis.

6. A method for evaluating the application of the Inula lactone of claim 1 in the preparation of a drug for treating diabetic wounds, characterized in that: The treatment assessment method of the model system The study included the following steps: constructing a model system and adding Inula lactone to the model system to systematically evaluate the effect of Inula lactone in the treatment of diabetic wounds.

7. The treatment assessment method according to claim 6, characterized in that: The model system includes a type 2 diabetic mouse model and a normal mouse model.

8. The treatment assessment method according to claim 7, characterized in that: Based on a type II diabetic mouse model, the dosage of the inula lactone was 10-20 mg / kg / day.

9. The treatment assessment method according to claim 8, characterized in that: High glucose combined with palmitic acid (PA) was added to the model system to simulate the wound environment of type 2 diabetes. Oxidative stress in each group was assessed using dichlorofluorescein-DA dye under a fluorescence microscope and flow cytometry. DCFH-DA dye is oxidized to green fluorescent substances by reactive oxygen species. The intensity of the green fluorescence was analyzed to determine the antioxidant capacity of Inula lactone. Lipid peroxidation fluorescent probes (C11(581 / 591)) were used to assess lipid oxidative stress in each group under a fluorescence microscope and flow cytometry. C11(581 / 591) changed color with the degree of lipid peroxidation. Analyzing the color change determined whether Inula lactone could prevent oxidative damage and effectively inhibit ferroptosis. Ferrous ion fluorescent probes (ferro orange) were used to assess iron overload in each group under a fluorescence microscope. Ferro orange dye produces red fluorescence after binding with iron ions to determine intracellular iron ion concentration, thus evaluating the role of Inula lactone in anti-oxidative stress and anti-ferroptosis.

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