Use of methylnaphthoquinone for the preparation of a medicament for the prevention and / or treatment of hypertrophic scars

Abstract

The application belongs to the technical field of medicines, and particularly relates to the use of methylbenzoquinone in the preparation of medicines for preventing and / or treating hyperplastic scars. The application first finds through experiments that methylbenzoquinone can promote the apoptosis of fibroblasts, reduce the expression of IL-8, IL-beta, Col I, Col III and other fibrosis-related genes, thereby effectively treating hyperplastic scars, which shows that methylbenzoquinone has the potential to be used as a medicine for treating hyperplastic scars, and provides more choices for clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of methylbenzoquinone in the preparation of medicaments for the prevention and / or treatment of hypertrophic scars. Background Technology

[0002] Hypertrophic scars (HS) are a common consequence of trauma, burns, and surgery. They are a fibroproliferative disorder resulting from abnormal healing after trauma, characterized by excessive deposition of extracellular matrix components and invasive proliferation of fibroblasts (Fb). The development of HS leads to impaired skin function and unsatisfactory cosmetic outcomes. Several treatment options are currently available, including surgical excision, laser therapy, and corticosteroid injections; however, the complex mechanisms of HS formation make these treatments unsatisfactory in clinical settings. Therefore, there is an urgent need to develop new treatment methods.

[0003] The pathogenesis of hepatic sclerosis (HS) is complex, but it is generally believed that the main causes are abnormal activation of fibrillary globulins (Fb) and the synthesis of extracellular matrix (ECM). Fb activation involves a transition from a relatively quiescent state to an activated state with excessive ECM production. This activation is transient during normal wound healing but persists during HS formation. Therefore, controlling targeted activated Fb and inhibiting its fibrotic behavior is the main strategy for controlling HS formation.

[0004] The traditional Yunnan folk medicine *Impatiens balsamina* has been shown to have significant anti-scarring effects. However, research on the material basis of *Impatiens balsamina* is still incomplete, and there are currently no reports on the active substances responsible for its anti-scarring effects. Methylbenzoquinone (2-Methylcyclohexa-2,5-diene-1,4-dione, CAS: 553-97-9) is one of the active ingredients of *Impatiens balsamina*. In the current technology, methylbenzoquinone is mainly used as a pigment, dye, and pharmaceutical intermediate. No reports have been found regarding the anti-scarring effects of methylbenzoquinone in the existing technology. Summary of the Invention

[0005] In view of the problems of the prior art, the present invention provides the use of methylbenzoquinone in the preparation of medicaments for the prevention and / or treatment of hypertrophic scars.

[0006] Use of methylbenzoquinone in the preparation of medicines for the prevention and / or treatment of hypertrophic scars.

[0007] Preferably, the drug is used to promote fibroblast apoptosis and inhibit the expression of fibrosis-related genes.

[0008] The present invention also provides the use of extracts of *Ligustrum lucidum* containing methylbenzoquinone in the preparation of medicaments for the prevention and / or treatment of hypertrophic scars.

[0009] Preferably, the drug is used to promote fibroblast apoptosis and inhibit the expression of fibrosis-related genes.

[0010] Preferably, the beak-tailed lute is *Blaps rynchopetera* Fairmaire.

[0011] The present invention also provides a medicament for the prevention and / or treatment of hypertrophic scars, which is a preparation made by adding pharmaceutically acceptable excipients or auxiliary ingredients to methylbenzoquinone or extracts of methylbenzoquinone-containing rhizomes.

[0012] Preferably, the drug is made from the following raw materials in parts by weight:

[0013] Methylbenzoquinone 0.5-1.5 parts

[0014] White petroleum jelly 29.5-30.5 servings

[0015] 9 parts liquid paraffin.

[0016] Preferably, the drug is used to promote fibroblast apoptosis and inhibit the expression of fibrosis-related genes.

[0017] Preferably, the beak-tailed lute is *Blaps rynchopetera* Fairmaire.

[0018] Preferably, the preparation is a solution, spray-dried powder, ointment, cream, gel, tincture, liniment, medicated paste, powder, oil, paste, plaster, film coating, or aerosol.

[0019] In this invention, the "beaked-tailed beetle" is Blaps rynchopetera Fairmaire, a medicinal insect belonging to the genus Blaps in the family Tenebrionidae of the order Coleoptera.

[0020] This invention has shown through experiments that methylbenzoquinone can promote fibroblast apoptosis and reduce the expression of fibrosis-related genes such as IL-8, IL-β, ColⅠ, and ColⅢ, thereby effectively treating hypertrophic scars. This indicates that methylbenzoquinone has the potential to be used as a treatment for hypertrophic scars, providing more options for clinical treatment.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following specific implementation manners in the form of embodiments will further elaborate on the above content of the present invention. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Brief Description of the Drawings

[0023] Figure 1 Gross view of the wound and HS tissue healed on the 49th day after surgery in Example 1;

[0024] Figure 2 Comparison of HE staining, Masson staining, VG staining, and Sirius red staining in each group in Example 1;

[0025] Figure 3 Comparison of scar hyperplasia indexes in each group in Example 1. Among them, compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001;

[0026] Figure 4 Comparison of Tunel staining and WB in each group in Example 1;

[0027] Figure 5 Comparison of the expression of pro-fibrotic genes in each group. Among them, compared with the model group, **P < 0.01, ***P < 0.001, ****P < 0.0001. Specific Embodiment

[0028] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0029] Example 1 Therapeutic Effect of Methylbenzoquinone on Hypertrophic Scar

[0030] I. Experimental Method

[0031] 1. Replication of the rabbit ear hypertrophic scar (HS) model

[0032] 10 ordinary Japanese white rabbits, weighing 3.0 - 3.3 kg, with a rabbit age of 3 - 4 months, regardless of gender, excluding pregnant rabbits, with both ears intact, raised in an ordinary environment, room temperature 18 - 26°C, indoor relative humidity 60 - 80%, free diet, and single-caged. All white rabbits are from the Experimental Animal Center of Kunming Medical University, with the experimental animal production license number SCXK(Yun)K2020 - 0004. This experiment has been approved by the Ethics Committee of Yunnan University of Traditional Chinese Medicine, with the ethics approval number R - 062022157.

[0033] Three rabbits were left as a control group, receiving no treatment and normal feeding. For the remaining rabbits, six sites were selected on each ear, with each site spaced at least 2 cm apart. The rabbits were restrained in a device, exposing their heads. Hair was shaved, and the skin was disinfected with alcohol swabs. 4 ml / kg of 25% urethane was slowly injected into the marginal ear vein. After successful anesthesia, a corneal trephine was used to drill circular wounds at the selected sites, 1 cm apart, reaching the cartilage surface. Skin was removed with forceps and scissors, and the perichondrium was removed with a blade and scissors, preserving the cartilage. Hemostasis was achieved. Bleeding and discharge were closely monitored for three days post-surgery, and the wounds were cleaned promptly. Afterward, the rabbits were allowed free access to water and food, and the wounds were allowed to heal naturally. Photos were taken on the day of medication and on days 7, 14, and 21 post-treatment to observe wound healing, scar hyperplasia, regression, and changes in texture and color.

[0034] 2. Experimental Design and Drug Management

[0035] For wounds that healed poorly or became infected and necrotic, the wounds were removed. Successful modeling was defined as the formation of scar tissue that was significantly raised above the normal skin but did not extend beyond the wound surface. Rabbits with successful modeling were divided into a model group and a methylbenzoquinone group. The model group was treated with a uniformly mixed ointment of white petrolatum and liquid paraffin in a ratio of 8:2. The methylbenzoquinone group was treated with a uniformly mixed lemon-yellow ointment of methylbenzoquinone powder (Chengdu Efa Biotechnology Co., Ltd., Purity: 98%, AFBL006), white petrolatum, and liquid paraffin in a ratio of 1:30:9. The ointment was applied three times daily, with 3-4 hour intervals between applications, using 0.1-0.3g each time, evenly covering the HS surface. Scar changes were observed weekly by taking photographs. After 21 days of continuous treatment, the rabbits died from an overdose of 20% urethane anesthesia. Rabbit ear HS tissue, including a small amount of normal skin at the HS margin and cartilage at the depth, was excised using a 12mm corneal trephine. Half of the total amount in each group was fixed with 10% neutral formaldehyde for pathological staining, while the other half was flash-frozen in liquid nitrogen and stored at -80°C for RT-qPCR and WB detection.

[0036] 3. Histological examination

[0037] The HS tissue was cut along its long axis, embedded in paraffin, and cut into 4 μm thick sections. Hematoxylin and eosin (H&E), Masson's trichrome, VG, Sirius red, and Tunel green staining were then performed. After fixation, Sirius red staining was observed under polarized light to infer the degree of fibrosis in the HS; Tunel staining was observed under a fluorescence microscope (Nikon Ecolipse C1, Japan) to determine the apoptosis status of fibrils (Fb); and other staining methods were observed under an upright optical microscope (Nikon Ecolipse E100, Japan) to observe the collagen content, distribution, and arrangement.

[0038] 4. Scar Hyperplasia Index (HI)

[0039] The scar hyperplasia index (HI) is calculated using H&E stained slides from Helicobacter pylori (HS) to quantify the degree of scar proliferation. The HI is calculated as follows: HE-stained slides are placed under a 40× light microscope, with the field of view including the highest point of the scar and the cartilage surface. The distance A from the highest point of the scar to the cartilage surface and the distance B from normal skin to the cartilage surface are measured using a microscopic measurement system. The ratio A / B is the HI.

[0040] 5. RNA extraction and RT-qPCR

[0041] RNA was isolated from tissues on postoperative day 49 using a rotor-stator homogenizer and the Tiangen Total RNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, Cat: DP49, Lot: X0713). 2 μl of total RNA was mixed with a Promega Reverse Transcription Kit (Promega, USA, 0004478178) to prepare a 20 μl total volume for reverse transcription. The transcription was performed at 42℃ for 15 min, 70℃ for 15 min, and 4℃ for 4 min, converting the reverse transcription to cDNA. After reverse transcription, 2 μl of cDNA template was mixed with a Promega Quantitative PCR Kit (Promega, USA, 0000531173) to prepare a 20 μl total volume for quantitative amplification. The reaction conditions of the quantitative PCR instrument (Archimed X6, Kunpeng (Xuzhou) Scientific Instruments Co., Ltd.) were set as follows: 95℃ for 15 s, 55℃ for 30 s, and 72℃ for 30 s for 40 cycles. The target genes for detection included the expression levels of Collagen I (COL I), Collagen III (COL I), IL-8, and IL-1β genes. The gene sequences are attached in Table 1. Each gene of interest was normalized to the housekeeping gene β-actin, and each was measured in triplicate. The fold change was compared relative to the control sample (using the ΔΔCq method).

[0042] Table 1 Primers used for quantitative polymerase chain reaction analysis

[0043]

[0044]

[0045] 6. Western blot

[0046] 6.1 Extraction of total protein

[0047] Following the grouping method used in animal experiments, tissue samples were washed twice with pre-chilled PBS. For every 100 μl compressed volume of tissue sample, 1 ml of RIPA (P0013C) supplemented with PMSF, 1.5 mol / L Tris HCl (pH 6.8) (ST768), or 1.5 mol / L Tris HCl (pH 8.8) (ST789) (Beyotime, Shanghai, China) was added. After complete lysis, the samples were centrifuged at 12000 rpm for 5 min at 4 °C. The supernatant was immediately transferred to a pre-chilled Eppendorf tube, which contained the extracted cellular proteins. These were then stored at -80 °C for later use. Protein quantification was performed using the BCA method. Finally, 5× loading buffer was added and the sample was boiled in a water bath for 10 min. The sample preparation was complete and the sample could be stored at -20 °C.

[0048] 6.2 Gel electrophoresis and membrane transfer

[0049] Depending on the molecular weight of the protein to be measured, prepare an 8% or 10% separating gel and a 4% compression gel, and pour the SDS-PAGE gel. After adding an appropriate amount of pre-chilled 1× electrophoresis buffer, add the previously biolabeled sample or total intracellular protein extract to the lanes (pre-stained protein marker and sample). Electrophoresis is performed at a constant voltage of 80V for about 30 minutes. After the sample enters the separating gel, the voltage is adjusted to 120V and electrophoresis is continued. When the target band reaches the appropriate position (refer to the position of the pre-stained protein marker), the electrophoresis is stopped. Cut the gel at the corresponding position according to the size of the target protein, and place the cut gel in transfer buffer. Discard the remaining gel. Cut a PVDF membrane (0.45μm) (IPFL85R) (Millipore, Schwalbach, Germany) to the size of the gel, activate it in methanol for 1 minute, and then soak it in transfer buffer. Filter paper is also soaked in transfer buffer for 15 minutes. Prepare the transfer "sandwich" according to the principle of PVDF membrane ≥ gel ≥ filter paper, and ensure that air bubbles are removed before starting constant voltage transfer. After the transfer was completed, the membrane was stained with Ponceau S for 5 minutes, followed by washing twice with TBST, and the proteins on the membrane were observed.

[0050] 6.3 Development

[0051] Wash the membrane with TBST to remove any residual liquid. Then, place the membrane in a resealable bag using a sealing machine, sealing three sides. Add primary antibody diluted to an appropriate concentration with TBST (primary antibody dilution concentrations are shown in Table 2), removing as many air bubbles as possible. Seal the bag and incubate overnight at 4°C. Cut open the resealable bag and wash the membrane three times with TBST, 10 min each time. Then, place the membrane in a sealed bag, add an appropriate amount of secondary antibody (1:5000), seal the bag, and incubate at room temperature for 1 h. Cut open the resealable bag and wash the membrane three times with TBST, 10 min each time. Mix equal volumes of chemiluminescence reagent A and solution B (ECL luminescence solution) (A38555) (Thermo Fisher Scientific, Pittsburgh, PA, USA); place the membrane protein side down in full contact with this mixture; acquire images using a GE luminescence imaging workstation.

[0052] Table 2 Antibodies and dilution

[0053]

[0054] 7. Statistical methods

[0055] Data was processed using GraphPad Prism 9.0 (GraphPad Software, Inc.), with the data being... The data are categorized as follows: If the data follows a normal distribution and the variance is uniform, multiple comparisons are performed using the ANOVA test and Bonferroni correction. If the variance is non-uniform, multiple comparisons are performed using Dunnett's T_3 within Welch's ANOVA test. If the data does not follow a normal distribution, multiple comparisons are performed using Dunn's test within the Kruskal-Wallis test. A p-value < 0.05 is considered statistically significant.

[0056] II. Experimental Results

[0057] 1. Methylbenzoquinone reduces the formation of H₂S.

[0058] Gross observation showed that the wound underwent complete re-epithelialization by day 21 post-surgery. No significant changes were observed in the control group. Compared to the control group, in the model group, localized hemangiomas (HS) were observed to be raised above the surrounding undamaged skin, firm in texture, and pale red in color. In the methylbenzoquinone group, apart from minor drug-induced pigmentation, the HS formed was flatter, softer, and less noticeable than in the model group. Figure 1 The experimental results indicate that 2.5% methylbenzoquinone ointment can improve the gross view of HS, making HS appear flatter.

[0059] 2. Effects of methylbenzoquinone on the histological characteristics of HS

[0060] On day 21 after medication, 10×H&E staining showed that the epidermis and dermis of the blank group were smooth and without hyperplasia. Compared with the blank group, the epidermis and dermis of the scar in the model group showed significant hyperplasia, which was higher than that of the adjacent uninjured skin. The height of the hyperplastic tissue after treatment with methylbenzoquinone was significantly reduced. 400×H&E staining showed that the collagen in the blank group was neatly arranged, with a small number of fibrillary cells (Fb) scattered in the tissue. The collagen in the model group was disordered, with a large number of inflammatory cells infiltrating the tissue, and Fb proliferating actively. The collagen in the methylbenzoquinone group was relatively disordered, with a small number of inflammatory cells infiltrating, and less Fb proliferation.

[0061] 400×Masson staining showed that the collagen in the blank group (blue) was neatly and orderly arranged; the collagen in the model group was sparsely and disordered, with more whorl nodules; and the collagen in the methylbenzoquinone group was sparsely arranged, with fewer whorl nodules.

[0062] 200×VG staining showed that the epidermis (yellow) in the blank group was the thinnest, and the collagen (red) was the most neatly arranged and the darkest. The epidermis in the model group was the thickest, and the collagen was arranged in a disordered and sparse manner. The epidermis in the methylbenzoquinone treatment group was thicker than the normal group but thinner than the model group, and the collagen arrangement was more disordered than the blank group but more orderly than the model group.

[0063] Sirius red staining under polarized light revealed that the control group was dominated by type III collagen (appearing green), with a small amount of type I collagen (appearing red or yellow) scattered throughout; the model group was dominated by type I collagen, with a small amount of type III collagen scattered throughout; in the methylbenzoquinone group, type I and type III collagen showed an alternating pattern, with type I collagen increasing compared to the control group but decreasing compared to the model group, and type III collagen significantly increasing compared to the model group but decreasing compared to the control group. Figure 2 ).

[0064] The above pathological staining also shows from a microscopic perspective that methylbenzoquinone can reduce the excessive proliferation of Fb, make the epidermis of HS thinner and closer to normal skin, make the collagen arrangement thinner and more orderly, reduce the ratio of type I collagen to type III collagen, and make HS softer and more elastic.

[0065] 3. Methylbenzoquinone reduces the HI of HS

[0066] The HI quantified the degree of HS proliferation. Compared with the control group, the HI was increased in both the model group and the treatment group; compared with the model group, the HI was significantly decreased in the methylbenzoquinone group. Figure 3 The above results indicate that methylbenzoquinone significantly reduced the thickness of the formed HS tissue when quantitatively assessing the thickness of the HS tissue, and the differences between groups were significant.

[0067] 4. Methylbenzoquinone promotes apoptosis in HS cells.

[0068] Tunel staining showed fewer apoptotic cells (shown as green) in the control and model groups, while the methylbenzoquinone group had more. Furthermore, Western blotting results showed that the model group had lower levels of the pro-apoptotic proteins Bax and Cleaved casepase 3 than the control group, and higher levels of the anti-apoptotic protein Bcl-2 than the control group. In contrast, the methylbenzoquinone group had significantly higher levels of the pro-apoptotic proteins Bax and Cleaved casepase 3 than the other two groups, and significantly lower levels of the anti-apoptotic protein Bcl-2 than the other two groups. Figure 4 ).

[0069] The above results indicate that methylbenzoquinone can promote Fb apoptosis.

[0070] 5. Effects of methylbenzoquinone on HS gene expression

[0071] Compared with the control group, the expression of genes promoting hemorrhage (HS), such as IL-8, IL-1β, ColⅠ, and ColⅢ, was increased in both the model group and the methylbenzoquinone group; compared with the model group, the expression of HS-promoting genes was significantly decreased in the methylbenzoquinone group. Figure 5 ).

[0072] The above results indicate that methylbenzoquinone reduces the expression of profibrotic genes such as IL-8, IL-β, ColⅠ, and ColⅢ.

[0073] The results of the above examples show that methylbenzoquinone can promote fibroblast apoptosis and reduce the expression of fibrosis-related genes such as IL-8, IL-β, ColⅠ, and ColⅢ, thereby effectively treating hypertrophic scars. Therefore, methylbenzoquinone has the potential to be used as a treatment for hypertrophic scars and has a promising application prospect.

Claims

1. Use of methylbenzoquinone as a single active ingredient in the preparation of medicaments for the prevention and / or treatment of hypertrophic scars.

2. A medicine for the prevention and / or treatment of hypertrophic scars, characterized in that, It is a formulation prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients, with methylbenzoquinone as the single active ingredient.

3. The drug according to claim 2, characterized in that, The drug is made from the following raw materials in parts by weight: Methylbenzoquinone 0.5-1.5 parts White petroleum jelly 29.5-30.5 servings 9 parts liquid paraffin.

4. The drug according to claim 2, characterized in that, The preparation is a solution, ointment, cream, gel, powder, oil, paste, plaster, or aerosol.

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