Application of lithospermic acid in preparing drugs for treating diabetic ulcers

Abstract

The present invention provides the application of lithospermic acid in the preparation of drugs for treating diabetic ulcers, belonging to the technical field of drugs for diabetic ulcers. The present invention first proposes that the traditional Chinese medicine monomer lithospermic acid has the effect of treating diabetic ulcers. By using a mouse diabetic ulcer model and the human keratinocyte cell line HaCaT, the effects of lithospermic acid on the healing indexes and the expression of inflammatory indexes of diabetic ulcers in mice and in vitro cells are studied. The results show that lithospermic acid has a significant therapeutic effect on diabetic ulcers, providing an important basis for the future clinical development of efficient strategies for treating diabetic ulcers and the preparation of drugs for treating diabetic ulcers.

Description

Technical Field

[0001] The invention belongs to the technical field of diabetic ulcer drugs, and in particular relates to application of lithospermic acid in preparing drugs for treating diabetic ulcers. Background Art

[0002] Diabetic ulcers are a serious complication of diabetes that often occur on the feet, hence the name diabetic foot. As one of the most common and intractable chronic complications of diabetes, diabetic foot has an incidence rate as high as 25%, with more than 15% of patients facing the risk of amputation or even death at any time. The treatment of diabetic ulcers requires multidisciplinary collaboration. In addition to adjusting blood sugar, blood lipids, and blood pressure, treatment must adhere to the following principles: anti-infection, timely revascularization, decompression to reduce trauma to the ulcer site, and wound treatment to promote healing. However, even with standard treatment measures such as debridement, dressings, decompression, anti-infection, and blood sugar regulation, the cure rate of diabetic ulcers can only reach 31% after 20 weeks of treatment. Therefore, finding an efficient and simple treatment method for diabetic ulcers is a key issue that needs to be urgently addressed in clinical practice.

[0003] Salvia miltiorrhiza (Danshen) is the dried root and rhizome of the Lamiaceae plant Salvia miltiorrhiza. It has the effects of promoting blood circulation and removing blood stasis, cooling blood and reducing swelling, and clearing the heart and relieving restlessness. Lithospermic acid is the main active ingredient in Salvia miltiorrhiza. In recent years, studies have shown that lithospermic acid has anti-inflammatory effects and inhibits uric acid formation in gout rat models. Lithospermic acid is widely found in Chinese medicinal materials such as Chuanxiong, Angelica sinensis, and Crocus sativus, is easy to extract, and is relatively cheap. Currently, there are no reports in the field regarding the correlation between lithospermic acid and the treatment of diabetic ulcers, and the development of new drugs for the treatment of diabetic ulcers is of great significance. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide the use of lithospermic acid in the preparation of a medicament for treating diabetic ulcers, wherein lithospermic acid has a significant therapeutic effect in treating diabetic ulcers.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of lithospermic acid in preparing medicine for treating diabetic ulcer.

[0007] Preferably, the lithospermic acid exerts its effect by promoting wound healing and re-epithelialization of diabetic ulcers.

[0008] Preferably, the lithospermic acid exerts its effect by promoting the proliferation and migration of human keratinocytes.

[0009] Preferably, the lithospermic acid exerts its effect by inhibiting the expression of inflammatory factors in diabetic ulcer wounds.

[0010] Preferably, the inflammatory factors include Il-6 and Il-1β.

[0011] The present invention also provides a drug for treating diabetic ulcers, and the drug uses lithospermic acid as the sole active ingredient.

[0012] Preferably, the drug is an external preparation.

[0013] Advantages of the present invention:

[0014] The present invention first proposes that the Chinese medicine monomer lithospermic acid has the effect of treating diabetic ulcers, and has achieved remarkable treatment effects, providing a basis for the future clinical development of high-efficiency strategies for treating diabetic ulcers.

[0015] The present invention uses a mouse diabetic ulcer model and the human keratinocyte cell line HaCaT to study the effects of lithospermic acid on the healing indexes and inflammatory index expressions of diabetic ulcers in mice and in vitro cells, providing important experimental basis for the preparation of drugs for treating diabetic ulcers. Description of the Drawings

[0016] Figure 1 Shows the effect of lithospermic acid on the wound healing of diabetic ulcer mice. Among them, Figure A shows the results of wound healing photography and the calculation results of wound healing rate, and Figure B shows the HE staining results;

[0017] Figure 2 Shows the effect of lithospermic acid on the proliferation and migration of HaCaT cells. Among them, Figure A shows the results of the CCK-8 experiment, and Figure B shows the results of the scratch experiment;

[0018] Figure 3 Shows the effect of lithospermic acid on the mRNA expressions of inflammatory factors Il-6 and Il-1β in HaCaT cells. Detailed Embodiments

[0019] The present invention provides the application of lithospermic acid in the preparation of drugs for treating diabetic ulcers.

[0020] In the present invention, the chemical structural formula of the lithospermic acid is:

[0021]

[0022] The molecular formula is C 27 H 22 O 12 .

[0023] The present invention has no special limitation on the specific source of lithospermic acid, which can be obtained by self-extraction and preparation or purchased commercially. In the present invention, the lithospermic acid preferably plays a role in promoting the healing and re-epithelialization of diabetic ulcer wounds, and more preferably plays a role in promoting the proliferation and migration of human keratinocytes. In the present invention, the lithospermic acid also plays a role in inhibiting the expression of inflammatory factors in diabetic ulcer wounds, and the inflammatory factors preferably include Il-6 and Il-1β.

[0024] The present invention also provides a drug for treating diabetic ulcers, and the drug uses lithospermic acid as the sole active ingredient.

[0025] In the present invention, the drug is preferably an external preparation, and the drug preferably further includes excipients commonly used in external preparations. The present invention has no special limitation on the types and specific sources of excipients commonly used in external preparations, and common excipients in the field of external preparations can be used.

[0026] The technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they cannot be understood as limiting the protection scope of the present invention.

[0027] In the following examples, unless otherwise specified, all are conventional methods.

[0028] In the following examples, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0029] Example 1

[0030] Effect of lithospermic acid on wound healing of diabetic ulcer mice

[0031] 1. Animals: C57 / BL male mice (6 - 7 weeks old), weighing 22 - 24 g.

[0032] Animal model: Diabetic group mice were fed a high-fat diet for 4 weeks. Except for the normal control group (4 mice), all mice were injected with streptozotocin (STZ) every other day and drank 5% glucose solution 4 hours later. After 7 days, the blood glucose level was measured. If the blood glucose concentration was higher than 300.6 mg / dl -1 , and the diabetic mice were monitored to have symptoms of polyuria, polyphagia, and polydipsia, it was considered that the diabetic model was successfully constructed. Mice with a weight loss of more than 20% or poor motor ability were excluded. The 12 successfully established diabetic mice were randomly divided into a diabetic group, a diabetic lithospermic acid treatment group, and a diabetic positive treatment group. Before making the ulcer model, the mice were anesthetized with isoflurane, and then 4 cm × 4 cm of hair was removed from the back, and 4 circular wounds with a width of 6 mm and a depth of 2 mm were made with a puncher. The experiment was carried out under sterile conditions.

[0033] 2. Grouping and treatment:

[0034] (1) Control group: Ulcer models were established without any drug treatment; (2) STZ group: Ulcer models were established without any drug treatment; (3) STZ + LA group: Lithospermic acid monomer was dissolved in ethanol solution at a concentration of 50 μg / ml, and the ulcer sites of mice were wet compress with a dosage of 50 μl / rat / day for 9 consecutive days; (4) STZ + rb-bFGF group: Becaplermin (rb-bFGF) at a concentration of 200 ng / ml was topically applied to the ulcer sites of mice with a dosage of 50 μl / rat / day for 9 consecutive days.

[0035] 3. Experimental methods:

[0036] (1) Wound healing analysis

[0037] Macroscopic observation: Photos were taken on the 1st, 3rd, 5th, 7th, and 9th days after drug treatment. The ulcer area of the wound surface was calculated by processing the photographed images using Image-J software. The wound closure was calculated using the following formula:

[0038] Wound healing rate (%) = (1 - width of treated wound / width of control wound) × 100, where the width of the control wound was the wound width at the time of model establishment on Day 0.

[0039] (2) Histological hematoxylin-eosin staining (HE staining)

[0040] Mice were sacrificed on the 5th and 9th days after induction. Skin specimens were taken and fixed in 4% neutral formaldehyde buffer for 24 h. Routine histological examination, paraffin embedding, sectioning, and HE staining were performed. The sections were observed under a digital slide scanner.

[0041] 4. Experimental results

[0042] Lithospermic acid can improve wound healing in diabetic ulcer mice

[0043] The effect of lithospermic acid on diabetic ulcer wound healing in vivo was observed by wound healing analysis and HE staining. The results are as Figure 1 shown. As can be seen from Figure 1 A, compared with the diabetic positive treatment group, the diabetic lithospermic acid treatment group had a more significant wound healing effect on the 5th, 7th, and 9th days after injury. The HE staining results ( Figure 1 B) showed that the wounds in the diabetic lithospermic acid treatment group were significantly reduced on the 5th and 9th days, and the effect was better than that of the diabetic positive treatment group. The results indicate that the wound healing of STZ-induced diabetic mice is slowed down, and lithospermic acid intervention can effectively promote diabetic wound healing and re-epithelialization, and the curative effect is more significant than that of the positive control becaplermin.

[0044] Example 2

[0045] Effect of lithospermic acid on the proliferation and migration of HaCaT cells in vitro

[0046] 1. Cell model: Human keratinocyte cell line HaCaT. Cells were cultured in DMEM-high glucose containing 10% fetal bovine serum and 1% streptomycin-penicillin solution, and incubated at a constant temperature of 37°C.

[0047] 2. Grouping:

[0048] (1) Normal group (NC); (2) Lithospermic acid treatment group (LA): HaCaT cells were intervened with lithospermic acid (LA) at a concentration of 5 μg / ml; (3) Positive control group (bFGF): HaCaT cells were intervened with the positive control drug basic fibroblast growth factor (bFGF) at a concentration of 20 ng / ml.

[0049] 3. Experimental methods:

[0050] (1) Cell Counting Kit-8 (CCK-8) assay

[0051] CCK-8 was used to evaluate the proliferation of HaCaT cells according to the manufacturer's instructions. Briefly, cells were seeded in 96-well plates at a density of 2000 cells / 100 μL in three replicates. At the designated time points, CCK-8 solution was added to detect the absorbance at 450 nm. The absorbance values were normalized to be consistent with those of the control cells. Data from 3 independent experiments were used as the mean standard deviation.

[0052] (2) Scratch assay

[0053] The cells in the logarithmic growth phase collected by digestion were diluted and seeded into 6-well plates at a cell density of 5×10 5 cells per well. Each experimental group had 3 replicate wells. When the cells were observed to grow as a monolayer adherent under the microscope, a 200 μl pipette tip and a ruler were used to make a vertical scratch above the 6-well plate. Then, the plate was washed several times with PBS to remove as many floating cells as possible. Subsequently, lithospermic acid and basic fibroblast growth factor (bFGF) were added for treatment, and the plate was placed in an incubator with 5% CO2 (37°C, relative humidity 95%) for culture. Photos were taken under the microscope at 0 h, 6 h, 12 h, and 24 h. The cell migration area was calculated using Image-J software, and the scratch area repair rate of each group was analyzed. The formula is as follows:

[0054] Scratch area repair rate = (Scratch area at 0 h - Scratch area) / Scratch area at 0 h × 100%

[0055] 4. Experimental results:

[0056] Lithospermic acid promotes the proliferation and migration of HaCaT cells

[0057] The CCK-8 results showed that the proliferation ability of the lithospermic acid group cells was significantly higher than that of the normal group and the positive control group at 48 h and 72 h. Figure 2 A). The scratch test showed that the migration ability of HaCaT cells was significantly enhanced after 12 h and 24 h of lithospermic acid treatment. Figure 2 B). The above results indicate that lithospermic acid can effectively promote the proliferation and migration ability of HaCaT cells, and its effect is better than that of bFGF.

[0058] Example 3

[0059] Effect of in vitro application of lithospermic acid on the expression of inflammatory factors Il-6 and Il-1β mRNA in HaCaT cells

[0060] 1. Cell model: Human keratinocyte cell line HaCaT.

[0061] 2. Grouping:

[0062] (1) Normal group (NC); (2) Disease group (MGO): HaCaT cells were intervened with methylglyoxal (MGO) to induce an in vitro inflammatory model of diabetic ulcers; (3) Lithospermic acid treatment group (MGO+LA): Treated with 5 μg / ml LA on the basis of the disease group; (4) Positive control group (MGO+bFGF): Treated with 20 ng / ml of the positive control drug bFGF on the basis of the disease group.

[0063] 3. Experimental method:

[0064] Real-time quantitative polymerase chain reaction (qPCR)

[0065] Total RNA was extracted from HaCaT cells using the standard TRIzol protocol. Then, the RNA was reverse transcribed into cDNA using a reverse transcription reagent (TAKARAPrimeScript RT Master Mix (Perfect Real Time) RR036A). Next, TB Green Premix Ex Taq II (Tli RNaseH Plus), Code No. RR820A was selected for Real Time PCR reaction. After the reaction, data were collected, and the 2 -ΔΔCT method was used to analyze the relevant quantitative data, and the fold change was expressed as the expression level of RNA relative to the blank control.

[0066] The detailed information of the primers is shown in Table 1 below:

[0067] Table 1 Primer sequences

[0068]

[0069] 4. Experimental results

[0070] Lithospermic acid inhibits the expression of inflammatory factors Il-6 and Il-1β mRNA in HaCaT cells

[0071] In this example, MGO was used to intervene in HaCaT cells to induce an in vitro inflammatory model of diabetic ulcers. The results showed that the expression of inflammatory factors Il-6 and Il-1β mRNA increased, while lithospermic acid could significantly reduce the inflammatory infiltration induced by MGO, and the effect was more significant than that of bFGF( Figure 3 ).

[0072] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of lithospermic acid in the preparation of a drug for treating diabetic ulcers, characterized in that, The lithospermic acid plays a role by promoting the healing and re-epithelialization of diabetic ulcer wounds; the lithospermic acid plays a role by promoting the proliferation and migration of human keratinocytes.

2. The application according to claim 1, wherein The lithospermic acid plays a role by inhibiting the expression of inflammatory factors in diabetic ulcer wounds.

3. The application according to claim 2, wherein The inflammatory factors include Il-6 and Il-1β.

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