Use of lithospermic acid in the manufacture of a drug for treating diabetic ulcers

Abstract

The present invention provides the use of lithospermic acid in the preparation of drugs for treating diabetic ulcers, and belongs to the technical field of drugs for treating diabetic ulcers.The present invention is the first to propose that lithospermic acid, a single substance of traditional Chinese medicine, has the effect of treating diabetic ulcers, and uses a mouse diabetic ulcer model and a human keratinocyte cell line, HaCaT, to study the effects of lithospermic acid on diabetic ulcer healing indexes and inflammation indexes in mice and in vitro cells, which provides important basis for the development of an effective treatment method for diabetic ulcers in clinical practice and the preparation of drugs for treating diabetic ulcers.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of diabetic ulcer medication, and particularly relates to the use of lithospermic acid in the preparation of diabetic ulcer medication. [Background technology]

[0002] Diabetic ulcers are a serious complication of diabetes and often occur on the patient's foot, hence the name diabetic foot. Diabetic foot is a common and difficult-to-treat chronic complication of diabetes, with a high incidence of 25%. Of these, more than 15% of patients face the risk of amputation or death at any given time. Diabetic ulcer treatment requires multidisciplinary collaboration and should be based on principles including controlling blood glucose, blood lipids, and blood pressure, anti-infection, timely revascularization, decompression to reduce wound size at the ulcer site, and wound care to promote healing. However, even with standard treatments such as wound cleaning, dressings, decompression, anti-infection, and blood glucose control, the healing rate for diabetic ulcers remains at only 31% after 20 weeks of treatment. Therefore, establishing an efficient and convenient diabetic ulcer treatment remains a significant clinical challenge.

[0003] Danshen is the dried root and rhizome of the mint family plant Danshen, and has the effects of activating blood, cooling blood, dissipating swelling, and clearing the heart and relieving stress. Lithospermic acid is the main active ingredient of Danshen, and recent studies have shown its anti-inflammatory and uric acid production inhibitory effects in rat models of gout. Lithospermic acid is also widely found in traditional Chinese medicines such as Cnidium Rhizome, Angelica Root, and saffron, and is easy to extract and relatively inexpensive. At present, there are no reports in the field regarding the relationship between lithospermic acid and the treatment of diabetic ulcers, and its use is considered to be of great significance in the development of new drugs for the treatment of diabetic ulcers. Summary of the Invention [Problem to be solved by the invention]

[0004] In view of the above background, the present invention aims to provide a use of lithospermic acid in the manufacture of a medicament for treating diabetic ulcers, and lithospermic acid has a significant effect in treating diabetic ulcers. [Means for solving the problem]

[0005] In order to achieve the above object of the invention, the present invention provides the following technical solutions. The present invention provides a use of lithospermic acid in the manufacture of a medicament for treating diabetic ulcers.

[0006] Preferably, the lithospermic acid exerts its effect by promoting healing and re-epithelialization of diabetic ulcer wounds.

[0007] Preferably, the lithospermic acid acts by promoting the proliferation and migration of human keratinocytes.

[0008] Preferably, the lithospermic acid exerts its effect by suppressing the expression of inflammatory factors at the wound surface of a diabetic ulcer.

[0009] Preferably, the inflammatory factors include Il-6 and Il-1β.

[0010] The present invention further provides a drug for treating diabetic ulcers, which contains lithospermic acid as the only active ingredient.

[0011] Preferably, the drug is in an external preparation. [Effects of the Invention]

[0012] This invention is the first to propose that lithospermic acid, a single component of traditional Chinese medicine, has the effect of treating diabetic ulcers, and has achieved significant therapeutic effects, providing a basis for the development of an effective treatment plan for diabetic ulcers in future clinical practice.

[0013] The present invention uses a mouse diabetic ulcer model and the human keratinocyte cell line HaCaT to study the effects of lithospermic acid on diabetic ulcer healing and inflammation indices in mice and in vitro cells, providing important experimental evidence for the development of drugs for the treatment of diabetic ulcers. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the effect of lithospermic acid on the wound healing status of diabetic ulcer mice, where FIG. A shows photographic results of wound healing and calculation results of wound healing rate, and FIG. B shows the results of HE staining. [Figure 2] FIG. 1 shows the effect of lithospermic acid on the proliferation and migration of HaCaT cells, where panel A shows the results of a CCK-8 experiment and panel B shows the results of a scratch experiment. [Figure 3] FIG. 1 shows the effect of lithospermic acid on the expression of inflammatory factor Il-6 and Il-1β mRNA in HaCaT cells. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a use of lithospermic acid in the manufacture of a medicament for treating diabetic ulcers.

[0016] In the present invention, the chemical structural formula of the lithospermic acid is as follows:

[0017] [ka]

[0018] The molecular formula is C 27 H 22 O 12 is.

[0019] The specific source of lithospermic acid in the present invention is not particularly limited, and it can be obtained by self-extraction or preparation, or it can be purchased as a commercially available product. In the present invention, the lithospermic acid preferably acts by promoting healing and re-epithelialization of diabetic ulcer wound surfaces, and more preferably, by promoting the proliferation and migration of human keratinocytes. In the present invention, the lithospermic acid acts by suppressing the expression of inflammatory factors in diabetic ulcer wound surfaces, and the inflammatory factors preferably include IL-6 and IL-1β.

[0020] The present invention further provides a drug for treating diabetic ulcers, which contains lithospermic acid as the only active ingredient.

[0021] In the present invention, the drug is preferably an external preparation, and the drug preferably further comprises auxiliary materials commonly used in external preparations. The present invention does not particularly limit the types and specific sources of auxiliary materials commonly used in external preparations, and common auxiliary materials for external preparations in the art may be used.

[0022] The technical solutions provided by the present invention will be described in detail below with reference to examples, which should not be construed as limiting the scope of protection of the present invention.

[0023] In the following examples, all procedures are conventional unless otherwise specified.

[0024] All materials and reagents used in the following examples are available from commercial sources unless otherwise noted. [Example]

[0025] Effect of lithospermic acid on wound healing in diabetic ulcer mice 1. Animals: C57 / BL male mice (6-7 weeks old), weighing 22-24g. Animal model: Mice in the diabetic group were fed a high-fat diet for 4 weeks. All mice, except for the normal control group (4 mice), were injected with streptozotocin (STZ) every other day and ingested a 5% glucose solution 4 hours after the injection. After 7 days, blood glucose levels were measured and the blood glucose concentration was 300.6 mg / dl. -1 The diabetic model was considered to have been successfully constructed if the diabetic mice exhibited symptoms of polyuria, polyphagia, and polydipsia. Mice with weight loss exceeding 20% ​​or reduced exercise capacity were excluded. The 12 diabetic mice were randomly divided into a diabetic group, a diabetic lithospermic acid treatment group, and a diabetic positive treatment group. Before constructing the ulcer model, mice were anesthetized with isoflurane, a 4 cm x 4 cm area of ​​hair removed from the back, and four circular wounds, each 6 mm in diameter and 2 mm deep, were created using a punch. Experiments were conducted under aseptic conditions.

[0026] 2. Grouping and Treatment (1) Normal group (Control): An ulcer model was created, but no drug treatment was administered. (2) Diabetic group (STZ): An ulcer model was created, but no drug treatment was administered. (3) Diabetic lithospermic acid treatment group (STZ+LA): Lithospermic acid monomer was dissolved in ethanol solution to a concentration of 50 μg / ml, and applied to the ulcer site of the mouse at a dose of 50 μl / mouse / day for 9 consecutive days. (4) Diabetic positive treatment group (STZ+rb-bFGF): Befgy (rb-bFGF) at a concentration of 200 ng / ml was applied topically to the ulcer site of the mouse at a dose of 50 μl / mouse / day for 9 consecutive days.

[0027] 3. Experimental Method (1) Wound healing analysis Overall observation: Photographs were taken on the 1st, 3rd, 5th, 7th, and 9th days after treatment, and the images were processed using Image-J software to calculate the ulcer area on the wound surface. The wound closure status was calculated using the following formula.

[0028] Wound healing rate (%) = (1 - treated wound width / control wound width) x 100, where the control wound width is the width of the wound created on Day 0.

[0029] (2) Histological hematoxylin-eosin staining (HE staining) Mice were sacrificed on days 5 and 9 after induction, and skin specimens were fixed in 4% neutral formalin buffer for 24 hours. Then, standard histological examination, paraffin embedding, sectioning, and HE staining were performed. The specimens were then viewed using a digital section scanner.

[0030] 4. Experimental Results Lithospermic acid can improve wound healing in diabetic ulcer mice. Wound healing analysis and HE staining were used to observe the effects of lithospermic acid on diabetic ulcer wound healing in vivo. The results are shown in Figure 1. Figure 1A shows that the diabetic lithospermic acid treatment group had a more pronounced wound healing effect on days 5, 7, and 9 after wounding compared with the diabetic control treatment group. HE staining results (Figure 1B) showed that the diabetic lithospermic acid treatment group had significantly reduced wound size on days 5 and 9, demonstrating superior efficacy compared with the diabetic control treatment group. These results indicate that although wound healing is delayed in STZ-induced diabetic mice, lithospermic acid treatment effectively promoted diabetic wound healing and re-epithelialization, demonstrating a more pronounced therapeutic effect than the positive control, VEGF. [Example]

[0031] The effect of lithospermic acid on the proliferation and migration of HaCaT cells in vitro 1. Cell model: Human keratinocyte cell line HaCaT. Cells were cultured at 37°C in DMEM-high sugar medium containing 10% fetal bovine serum and 1% streptomycin-penicillin solution.

[0032] 2. Grouping: (1) Normal group (NC), (2) Lithospermic acid treatment group (LA): HaCaT cells were treated with lithospermic acid (LA) at a concentration of 5 μg / ml, and (3) Positive control group (bFGF): HaCaT cells were treated with basic fibroblast growth factor (bFGF), a positive control drug, at a concentration of 20 ng / ml.

[0033] 3. Experimental Method: (1) Cell Counting Kit-8 (CCK-8) detection CCK-8 was used to assess HaCaT cell proliferation according to the manufacturer's instructions. Briefly, cells were seeded into 96-well plates at a density of 2000 cells / 100 μL in triplicate. After seeding, CCK-8 solution was added at the indicated time points, and absorbance was measured at 450 nm. Absorbance values ​​were normalized to match those of control cells. Data from three independent experiments are presented as mean standard deviation.

[0034] (2) Scratch experiment Dissociated and collected cells in the logarithmic growth phase were diluted to a cell density of 5 × 10 5 Cells were seeded into 6-well plates at 1000 cells / well, with three wells per group. After observing the cells growing in a monolayer under a microscope, a vertical scratch was made at the top of the 6-well plate using a pre-sterilized 200 μL pipette tip and a ruler. The cells were then washed multiple times with PBS to remove as many floating cells as possible. The cells were then treated with lithospermic acid or basic fibroblast growth factor (bFGF), respectively, and cultured in a culture chamber at 5% CO2 (37°C, 95% relative humidity). Images were taken under a microscope after 0, 6, 12, and 24 hours. The cell migration area was calculated using Image-J software, and the scratch repair rate for each group was analyzed. The formula is as follows:

[0035] Scratch area repair rate = (0h scratch area - scratch area) / 0h scratch area x 100%

[0036] 4. Experimental results: Lithospermic acid promotes proliferation and migration of HaCaT cells The CCK-8 results showed that the proliferation ability of cells in the lithospermic acid group was significantly higher than that of the normal group and the positive control group at 48 and 72 hours (Figure 2A). In the scratch experiment, the migration ability of HaCaT cells was significantly enhanced at 12 and 24 hours after lithospermic acid treatment (Figure 2B). These results indicate that lithospermic acid effectively promotes the proliferation and migration ability of HaCaT cells, and its effect is superior to that of bFGF. [Example]

[0037] In vitro effects of lithospermic acid on the expression of inflammatory factors Il-6 and Il-1.BETA. mRNA in HaCaT cells 1. Cell model: human keratinocyte cell line HaCaT.

[0038] 2. Grouping: (1) Normal group (NC), (2) Disease group (MGO): Methylglyoxal (MGO) was used to induce an ex vivo diabetic ulcer inflammation model in HaCaT cells, (3) Lithospermic acid treatment group (MGO + LA): The same as the disease group, treated with 5 μg / ml of LA, and (4) Positive control group (MGO + bFGF): The same as the disease group, treated with 20 ng / ml of the positive control drug bFGF.

[0039] 3. Experimental Method: Real-time quantitative polymerase chain reaction (qPCR) Total RNA was extracted from HaCaT cells using the standard TRIzol method. The RNA was then reverse transcribed into cDNA using a reverse transcription reagent (TAKARA PrimeScript RT Master Mix (Perfect Real Time) RR036A). Real-time PCR was then performed using TB Green Premix Ex Taq II (Tli RNase H Plus) (Code No. RR820A). Data were collected after the reaction was completed. -ΔΔCT The relevant quantitative data were analyzed using the method and expressed as fold difference and RNA expression level relative to the blank control. Detailed information on the primers is shown in Table 1 below.

[0040] [Table 1]

[0041] 4. Experimental results: Lithospermic acid suppresses the expression of inflammatory factors Il-6 and Il-1β mRNA in HaCaT cells In this study, MGO was used to induce an in vitro diabetic ulcer inflammation model in HaCaT cells. The results showed that the expression of inflammatory factors Il-6 and Il-1β mRNA increased, while lithospermic acid significantly reduced the MGO-induced inflammatory infiltration, with a more pronounced effect than bFGF (Figure 3).

[0042] The above is only a preferred embodiment of the present invention, and those skilled in the art can make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

Claims

1. Use of lithospermic acid in the manufacture of a drug for treating diabetic ulcers.

2. The use according to claim 1, characterized in that the lithospermic acid exerts its effect by promoting healing and re-epithelialization of diabetic ulcer wound surfaces.

3. The use according to claim 1, characterized in that the lithospermic acid exerts its effect by promoting the proliferation and migration of human keratinocytes.

4. The use according to claim 1, characterized in that the lithospermic acid exerts its effect by suppressing the expression of inflammatory factors at the surface of diabetic ulcer wounds.

5. The use according to claim 4, characterized in that the inflammatory factors include Il-6 and Il-1β.

6. A drug for treating diabetic ulcers, characterized in that said drug contains lithospermic acid as the only active ingredient.

7. The drug according to claim 6, characterized in that the drug is an external preparation.

Images