Application of vanillic acid in preparation of medicine for treating diabetic ulcer

Abstract

The invention relates to the technical field of medicines, in particular to application of vanillic acid in preparation of a medicine for treating diabetic ulcer. The invention proposes that the vanillic acid has the effect of treating the diabetic ulcer for the first time, and by adopting a mouse diabetic ulcer model and a human skin fibroblast line HSF, the influence of the vanillic acid on the healing and ROS expression of a diabetic ulcer model mouse and the effect of the vanillic acid on the healing index and inflammatory response of the diabetic ulcer on in-vitro cells are researched. The result shows that the vanillic acid has a remarkable treatment effect on the diabetic ulcer, and an important basis is provided for clinically developing an efficient strategy for treating the diabetic ulcer and preparing the medicine for treating the diabetic ulcer in the future.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, in particular to application of vanillic acid in preparing medicines for treating diabetic ulcers. Background Art

[0002] Diabetic ulcers are common chronic complications of diabetes, often occurring in the patient's feet, so they are also called diabetic foot. Approximately 18.6 million people are affected by diabetic ulcers each year worldwide, with a probability of lower limb amputation of up to 80%, and the risk of death also increases. The mortality rate of diabetic patients without ulcers is about 18.2%, while the mortality rate of patients with diabetic ulcers increases to about 23.1%. Currently, surgical debridement, relieving ulcer pressure, and treating lower limb ischemia and foot infection are the main treatments for diabetic foot ulcers. However, only about 30%-40% of diabetic foot ulcers can heal within 12 weeks. Even if healed, the recurrence rate is high, estimated to be 42% at 1 year and 65% at 5 years (ARMSTRONG DG,TAN TW,BOULTON AJ M,etal.Diabetic Foot Ulcers:A Review[J].Jama,2023,330(1):62-75.FIFE CE,ECKERT KA,CARTER M J.Publicly Reported Wound Healing Rates:The Fantasy and theReality[J].Advances in wound care,2018,7(3):77-94.ARMSTRONG DG,BOULTON AJ M,BUS S A.Diabetic Foot Ulcers and Their Recurrence[J].The New England journal of medicine,2017,376(24):2367-75.) Therefore, finding an efficient and simple treatment method for diabetic ulcers is a key issue that needs to be urgently solved in clinical practice.

[0003] The specific mechanism of the difficulty in healing diabetic ulcers is still unclear, but fibroblasts play a key role in the proliferation and remodeling stages of diabetic wound healing. In the proliferation stage, fibroblasts can promote wound closure, matrix deposition and angiogenesis, and drive wound contraction by producing extracellular matrix (ECM) proteins and differentiating into myofibroblasts. In the remodeling stage, fibroblasts are the main cell type responsible for wound ECM remodeling. They replace the initial fibrin clot with hyaluronic acid, fibronectin and proteoglycans, and form mature collagen fibers in the later repair (WILKINSON HN, HARDMAN MJ. Wound healing: cellular mechanisms and pathological outcomes [J]. Open biology, 2020, 10 (9): 200223. LI J, CHEN J, KIRSNER R. Pathophysiology of acute wound healing [J]. Clinics in dermatology, 2007, 25 (1): 9-18. DARBY IA, LAVERDET B, BONTé F, et al. Fibroblasts and myofibroblasts in wound healing [J]. Clinical, cosmetic and investigational dermatology, 2014, 7: 301-11.). Therefore, promoting the proliferation of fibroblasts is crucial for the healing of diabetic wounds.

[0004] Vanillic acid is an antioxidant extracted from herbs, fruits, whole grains, juices, beer and wine, especially from the root of Angelica sinensis. The extraction process of high-purity vanillic acid has been maturely applied in industry, achieving mass production in a short period of time. As a phenolic compound, vanillic acid has multiple effects such as antibacterial, anti-inflammatory and antioxidant. It has been shown to block proinflammatory cytokines, inhibit inflammatory cascades, and reduce the expression of oxidation-related markers. In addition, vanillic acid has been shown to significantly inhibit the expression of multiple signaling pathways such as AMPK, NF-κB and JAK / STAT (LASHGARI NA, ROUDSARI NM, MOMTAZ S, et al. Regulatory Mechanisms of Vanillic Acid in Cardiovascular Diseases: A Review [J]. Current medicinal chemistry, 2023, 30 (22): 2562-76.).

[0005] Existing studies have shown that vanillic acid shows significant potential in the treatment of various diseases. Vanillic acid exhibits anti-tumor effects by activating the host stimulator of interferon genes (STING). Vanillic acid can alleviate liver fibrosis by inhibiting the MIF / CD74 signaling pathway. In addition, vanillic acid also reduces cardiotoxicity and myocardial ischemia-reperfusion injury by reducing the production of ROS and improving mitochondrial function. In an obese mouse model, vanillic acid reduces adipogenesis and body adipose tissue weight by activating AMPK (ZHU M, TANG X, ZHU Z, et al. STING activation in macrophages by vanillic acid exhibits antineoplastic potential[J]. Biochemical pharmacology, 2023, 213: 115618. QIN L, TAN J, LV X, et al. Vanillic acid alleviates liver fibrosis through inhibiting autophagy in hepatic stellate cells via the MIF / CD74 signaling pathway[J]. Biomedicine&pharmacotherapy=Biomedecine&pharmacotherapie, 2023, 168: 115673. YALAMEHA B, NEJABATI HR, NOURIM. Cardioprotective potential of vanillic acid[J]. Clinical and experimentalpharmacology&physiology, 2023, 50(3): 193-204. SHEKARI S,KHONSHA F,RAHMATI-YAMCHIM,et al.Vanillic Acid and Non-Alcoholic Fatty Liver Disease:A Focus on AMPKin Adipose and Liver Tissues[J].Current pharmaceutical design,2021,27(46):4686-92.).

[0006] However, no studies have investigated the relevance of vanillic acid in the treatment of diabetic ulcers, and the development of new drugs for the treatment of diabetic ulcers is of great clinical significance. Summary of the invention

[0007] The purpose of the present invention is to provide a new use of vanillic acid in treating diabetic ulcers.

[0008] The first aspect of the present invention provides the use of vanillic acid in preparing a drug for treating diabetic ulcers.

[0009] Furthermore, the molecular formula of the vanillic acid is C8H8O4, and the chemical structure is shown in Formula I below:

[0010]

[0011] The present invention has no particular limitation on the specific source of vanillic acid, which can be obtained by self-extraction and preparation, or purchased from the market.

[0012] Furthermore, in the drug, the vanillic acid plays a role by promoting the healing and re-epithelialization of diabetic ulcer wounds, reducing the expression of ROS in diabetic ulcer wounds, promoting the proliferation of human skin fibroblasts, and reducing inflammatory responses.

[0013] Furthermore, in the application, vanillic acid is used in the preparation of a drug for promoting wound healing and re-epithelialization of diabetic ulcers.

[0014] Furthermore, in the application, vanillic acid is used in the preparation of a drug for promoting the proliferation of human skin fibroblasts.

[0015] Furthermore, in the application, vanillic acid is used in the preparation of a drug for inhibiting inflammatory response in human skin fibroblasts.

[0016] Furthermore, in the application, vanillic acid is used in the preparation of a drug for reducing the expression of ROS in diabetic ulcer wounds.

[0017] In a second aspect, the present invention provides a drug for treating diabetic ulcers, wherein the drug contains vanillic acid as the only active ingredient.

[0018] Furthermore, the medicine is an external preparation, and further, the medicine also includes excipients commonly used in external preparations.

[0019] The present invention has no particular limitation on the types and specific sources of the commonly used auxiliary materials for external preparations, and the common auxiliary materials for external preparations in the art can be used.

[0020] Furthermore, the effective concentration of vanillic acid in the drug is 1 mM in vivo and 20 μM in vitro.

[0021] The present invention has the advantages that:

[0022] 1. The present invention is the first to discover that the Chinese medicine monomer vanillic acid has significant therapeutic effects in the treatment of diabetic ulcers, providing a solid basis for the future clinical development of efficient strategies for the treatment of diabetic ulcers.

[0023] 2. The present invention uses a mouse diabetic ulcer model and a human skin fibroblast cell line HSF to study the effects of vanillic acid on the healing and ROS expression of diabetic ulcer model mice, as well as the effects on diabetic ulcer healing indicators and inflammatory responses in cells in vitro. The results show that vanillic acid has a significant therapeutic effect on diabetic ulcers, which provides an important basis for the future clinical development of efficient strategies for the treatment of diabetic ulcers and the preparation of drugs for the treatment of diabetic ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of vanillic acid on wound healing in diabetic ulcer mice, where Figure A shows the photographic results of wound healing and the calculation results of wound healing rate; Figure B shows the HE staining results and the HE quantitative calculation results, scale bar = 1 mm;

[0025] Figure 2 The effect of vanillic acid on ROS expression in the wounds of diabetic ulcer mice, where the left picture shows the results of ROS detection by fluorescence microscopy in the wound surface, and the right picture shows the quantitative calculation results of ROS detection by fluorescence microscopy in the wound surface;

[0026] Figure 3 The effect of vanillic acid on HSF cell proliferation;

[0027] Figure 4 Effect of vanillic acid on inflammatory factors IL-1β and IL-6 in HSF cells. DETAILED DESCRIPTION

[0028] The specific embodiments provided by the present invention are described in detail below in conjunction with the examples and drawings. In the following examples, unless otherwise specified, all are conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0029] Example 1: Effect of vanillic acid on wound healing in diabetic ulcer mice

[0030] 1. Animals: Animal experiments used m / m control mice and db / db diabetic mice, male, 8 weeks old, provided by the Experimental Animal Center of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine Affiliated to Shanghai University of Traditional Chinese Medicine, and purchased from Jiangsu GemPharmatech Co., Ltd. All animals were kept at a standard temperature of 23±2°C, with SPF grade 12 h light / 12 h dark, and the animals had free access to high-fat or standard diets and water.

[0031] Animal model: 12 db / db diabetic mice were randomly divided into a diabetic group, a diabetic vanillic acid treatment group, and a diabetic positive treatment group. The hair on the back of the m / m normal control and db / db diabetic mice was shaved, and the shaved area was about 4×4cm; before the ulcer model was established, the mice were anesthetized with isoflurane, and then a wound model with an area of ​​6×6mm was created using a punch, with 4 wounds on the back of each mouse, and the experiment was carried out under sterile conditions.

[0032] 2. Grouping and treatment:

[0033] (1) Normal group (m / m): ulcer model was established without any drug treatment; (2) Diabetes group (db / db): ulcer model was established without any drug treatment; (3) Diabetes vanillic acid treatment group (db / db+VA): vanillic acid monomer was dissolved in a solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline) at a concentration of 1 mM, and applied to the mouse ulcer at a dose of 80 ul / mouse / day for 9 consecutive days; (4) Diabetes positive treatment group (db / db+rb-bFGF): bovine basic fibroblast growth factor topical solution (specification: 63000 IU / bottle) was applied topically to the mouse ulcer at a dose of 80 μl / mouse / day for 9 consecutive days.

[0034] 3. Experimental methods:

[0035] (1) Wound healing analysis

[0036] Macroscopic observation: Pictures were taken on the 3rd, 5th, 7th and 9th days after treatment, and the pictures were processed with Image-J software to calculate the wound ulcer area. The wound closure was calculated using the following formula:

[0037] Wound healing rate (%) = [(initial wound area - residual wound area) / initial wound area] × 100%

[0038] (2) Histological hematoxylin-eosin staining (HE staining)

[0039] The mice were killed on the 9th day after induction, and the skin specimens were fixed in 4% neutral formaldehyde buffer for 24 hours. Routine histological examination, paraffin embedding, sectioning, HE staining were performed, and the sections were observed under a digital section scanner.

[0040] 4. Experimental Results

[0041] Vanillic acid improves wound healing in mice with diabetic ulcers.

[0042] Wound healing analysis and HE staining were used to observe the effect of vanillic acid on diabetic ulcer wound healing in vivo. Figure 1As shown in A, compared with the diabetic ulcer group, the diabetic vanillic acid treatment group had a more significant wound healing effect on the 9th day after injury, and the effect was better than that of the positive treatment group. HE staining results ( Figure 1 B) shows that on day 9, the wound of the diabetic vanillic acid treatment group was significantly smaller than that of the diabetic ulcer group, and the effect was better than that of the diabetic positive treatment group. This result shows that the wound healing of db / db diabetic mice is slowed down, and vanillic acid intervention can effectively promote the healing and re-epithelialization of diabetic wounds, and the effect is more significant than that of the positive treatment group.

[0043] Example 2: Effect of vanillic acid on ROS expression in the wound surface of diabetic ulcer mice

[0044] 1. Animal model: m / m control mice and db / db diabetic mice.

[0045] 2. Grouping and treatment:

[0046] (1) Normal group (m / m): ulcer model was established without any drug treatment; (2) Diabetes group (db / db): ulcer model was established without any drug treatment; (3) Diabetes vanillic acid treatment group (db / db+VA): vanillic acid monomer was dissolved in a solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline) at a concentration of 1 mM, and applied to the mouse ulcer at a dose of 80 ul / mouse / day for 9 consecutive days; (4) Diabetes positive treatment group (db / db+rb-bFGF): bovine basic fibroblast growth factor topical solution at a concentration of 200 ng / ml (specification: 63000 IU / bottle) was applied topically to the mouse ulcer at a dose of 80 μl / mouse / day for 9 consecutive days.

[0047] 3. Experimental methods:

[0048] (1) Fluorescence staining and semi-quantitative analysis of ROS in wound surface

[0049] On the 9th day after induction, mice were killed, wound skin specimens were taken, and the expression of ROS in the wound was detected using a dihydroethidium ROS detection kit. 200 μl of cleaning solution was added to 10 μm frozen sections for 5 min, and the cleaning solution was removed; 50 μM dihydroethidium fluorescent probe was diluted 1000 times with PBS, and then added to the frozen sections, protected from light, and the wet box was placed in a 37°C water bath for 30 min; the dihydroethidium fluorescent probe was removed, washed 3 times with PBS, counterstained with DAPI, and washed 3 times with PBS; the sections were sealed with anti-fluorescence quenching sealing agent; ROS was detected under a fluorescence microscope. The mean fluorescence intensity of the fluorescence images was semi-quantitatively analyzed using Image-J software, and the mean fluorescence intensity was calculated using the following formula:

[0050] Average fluorescence intensity = total fluorescence intensity of the region / area of ​​the region

[0051] 4. Experimental results:

[0052] Vanillic acid can reduce the expression of ROS in the wound surface of diabetic ulcer mice.

[0053] The dihydroethidium ROS detection kit was used to detect the ROS content and observe the effect of vanillic acid on the expression of ROS in the wound surface of diabetic ulcer mice. Figure 2 As shown in the figure, compared with the diabetic ulcer group, the ROS content in the diabetic vanillic acid treatment group was significantly reduced and lower than that in the diabetic positive treatment group. This result shows that the expression of ROS in the wound of db / db diabetic mice is abnormally increased, and vanillic acid intervention can effectively reduce the expression of ROS in diabetic wounds, and the effect is more significant than that of the positive control Beifuji.

[0054] Example 3: Effect of in vitro application of vanillic acid on the proliferation of human skin fibroblast cell line HSF cells

[0055] 1. Cell model:

[0056] Human skin fibroblast cell line HSF. The cells were cultured in fibroblast medium containing 10% fetal bovine serum and 1% streptomycin-penicillin solution at 37°C.

[0057] 2. Grouping:

[0058] (1) Normal group (NC); (2) Vanillic acid treatment group (VA): HSF cells were treated with different concentrations of vanillic acid (VA).

[0059] 3. Experimental methods:

[0060] The vanillic acid stock solution was diluted in fibroblast culture medium with a final concentration gradient of 0, 2, 5, 10, 20, and 50 μM. After 48 hours, the proliferation of HSF cells was detected using a cell counting kit-8 (CCK-8), and the cell viability was calculated using the following formula:

[0061] Cell viability (%) = (average OD value of the experimental group - average OD value of the blank control) / (average OD value of the control group - average OD value of the blank control) × 100%

[0062] Briefly, cells were seeded in 96-well plates at a density of 5000 cells / 100 μL in three batches, and CCK-8 solution was added 48 hours after drug intervention to detect the absorbance at 450 nm. The absorbance values ​​were normalized to be consistent with the absorbance values ​​of control cells. The data of three independent experiments were used as the mean standard deviation.

[0063] 4. Experimental results:

[0064] Vanillic acid can promote the proliferation of HSF cells.

[0065] CCK-8 was used to detect the effect of vanillic acid at different concentrations on the proliferation activity of HSF cells. Figure 3 ). It was found that vanillic acid at in vitro concentrations of 5μM, 10μM, 20μM and 50μM could significantly promote HSF cell proliferation, among which 20μM and 50μM were the most significant, and the therapeutic effects were comparable.

[0066] Example 4: Effect of in vitro application of vanillic acid on the inflammatory response of human skin fibroblast cell line HSF cells

[0067] 1. Cell model:

[0068] Human skin fibroblast cell line HSF. The cells were cultured in fibroblast medium containing 10% fetal bovine serum and 1% streptomycin-penicillin solution at 37°C.

[0069] 2. Grouping:

[0070] (1) Normal group (NC); (2) Disease group (MGO): Methylglyoxal (MGO) was used to intervene in HaCaT cells to induce an in vitro inflammatory model of diabetic ulcer; (3) Vanillic acid treatment group (MGO+VA): The disease group was treated with 20 μM VA; (4) Positive control group (MGO+bFGF): The disease group was treated with 20 ng / ml of the positive control drug bFGF.

[0071] 3. Experimental methods:

[0072] Real-time quantitative polymerase chain reaction (qPCR)

[0073] Total RNA was extracted from HaCaT cells using the standard TRIzol protocol. RNA was then reverse transcribed into cDNA using a reverse transcription reagent (TAKARA PrimeScript RT Master Mix (Perfect Real Time) RR036A). Then, 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 relevant quantitative data were analyzed using the 2-ΔΔCT method, and the difference fold and blank control were expressed as RNA expression levels.

[0074] The detailed information of the primers is shown in Table 1 below:

[0075] Table 1 Primer sequences

[0076]

[0077] 4. Experimental Results

[0078] Vanillic acid inhibits the mRNA expression of inflammatory factors IL-6 and IL-1β in HSF cells

[0079] MGO was used to intervene HSF cells to induce an in vitro inflammatory model of diabetic ulcer. The results showed that the mRNA expression of inflammatory factors IL-6 and IL-1β increased, while vanillic acid could significantly reduce the inflammatory infiltration induced by MGO, and the effect was more significant than that of bFGF ( Figure 4 ).

[0080] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of vanillic acid in the preparation of drugs for the treatment of diabetic ulcers.

2. The use according to claim 1, characterized in that: Application of vanillic acid in the preparation of drugs for promoting wound healing and re-epithelialization of diabetic ulcers.

3. The use according to claim 1, characterized in that: Application of vanillic acid in preparing medicine for promoting proliferation of human skin fibroblasts.

4. The use according to claim 1, characterized in that: Application of vanillic acid in the preparation of drugs for inhibiting inflammatory response in human skin fibroblasts.

5. The use according to claim 1, characterized in that: Application of vanillic acid in the preparation of drugs for reducing ROS expression in diabetic ulcer wounds.

6. A drug for treating diabetic ulcer, characterized in that: The drug contains vanillic acid as the only active ingredient.

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

8. The drug according to claim 7, characterized in that The medicine also includes auxiliary materials commonly used in external preparations.

9. The drug according to claim 6, characterized in that The effective concentration of vanillic acid in the drug is 1 mM in vivo and 20 μM in vitro.

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