Use of scutellarein in the preparation of a medicament for treating burns and scalds

Abstract

The present application relates to the application of scutellarein in the preparation of drugs for treating burns and scalds, and belongs to the technical field of biological medicine. The present application discloses that scutellarein (5 g / kg) reduces the growth of bacteria on the burn (scald) wound by inhibiting the production and release of inflammatory mediators to reduce the inflammatory response of the wound; at the same time, the protein and gene expression levels of VEGF-A and TGF-beta1 are up-regulated, so that the expression peak is advanced, thereby shortening the time of exfoliation and hair growth of a small pig, and improving the healing rate of the wound. Scutellarein (5 g / kg) has obvious therapeutic effect on burns (scalds), and even is superior to the curative effect of 1% sulfadiazine silver (10 g / kg), which is a first-line drug in the clinic, and can be used for preparing a prophylactic and / or therapeutic drug for burns (scalds).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of ligustrazine in the preparation of drugs for treating burns and scalds. Background Technology

[0002] Burns, as an acute skin injury, are clinically predominantly small to medium-sized, with second-degree burns being the most common type. Modern burn medicine treats burn patients primarily with topical antibacterial drugs such as silver sulfadiazine, but these can worsen wound damage, resulting in a less than optimistic prognosis. Furthermore, numerous studies have found that traditional Chinese medicine and compound preparations have multiple targets and diverse mechanisms of action, which can compensate for many shortcomings of chemical drugs; however, research on the safety and efficacy of most traditional Chinese medicinal materials is still insufficient. With the deepening of basic research on burn pathology, how to effectively intervene and treat burns with topical medications has become an urgent problem to be solved by the medical community. Therefore, from both basic research and clinical application perspectives, it is essential and urgent to find topical medications with definite efficacy and fewer adverse reactions for the intervention and treatment of burns.

[0003] *Erigeron breviscapus* (Vant.) Hand.-Mazz., a perennial herb belonging to the genus *Erigeron* of the family Compositeaceae, is an important characteristic medicinal plant in Yunnan Province, accounting for over 90% of the national distribution. Erigeron breviscapus contains 4,5,6-trihydroxyflavone-7-glucuronide, a key active ingredient in this Yunnan specialty plant. Figure 1 Modern pharmacological studies have shown that scutellarin has the following effects: (1) anti-inflammatory and antioxidant effects: Scutellarin has anti-inflammatory activity, which may be related to its direct inhibition of Src enzyme activity.

[0004] In addition, it can inhibit collagen-induced arthritis, and its mechanism of action is to inhibit the TLR4 / NF-κB signaling pathway, etc. In addition, scutellarin has the effects of scavenging free radicals and antioxidation. (2) Neuroprotective effect: It mediates its neuroprotective effect by inhibiting the inflammatory activation of microglia; it protects against hypoxic-ischemic brain nerve injury by enhancing antioxidant defense capacity, etc.; (3) Cardiovascular and cerebrovascular protective effect: Scutellarin has a good protective effect against cerebral ischemia-reperfusion injury, and its mechanism of action may be to reduce hippocampal cell apoptosis, inhibit caspase-3 protein expression, upregulate Nrf2 / HO-1 protein expression, and improve tissue antioxidant capacity; (4) Anti-cancer effect, etc.

[0005] The healing process of burn wounds involves the joint participation of various inflammatory cells, growth factors, and extracellular matrix components, including burn-related growth factors such as vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1). TGF-β1 is a secreted polypeptide signaling molecule that regulates cell proliferation, differentiation, adhesion, migration, and apoptosis. In skin tissue homeostasis, the TGF-β1-mediated signaling pathway is crucial for effective wound repair and successful epithelialization. Among all growth factors, VEGF is considered the most effective in wound angiogenesis, especially in the early stages of wound healing, promoting endothelial cell migration and proliferation, and accelerating wound healing. Studies have confirmed that VEGF-A and TGF-β1, along with their receptor genes, participate as positive factors in the wound repair process, promoting scar formation, altering the pace of wound healing, and controlling fibrosis formation, playing a vital role in maintaining skin homeostasis.

[0006] In summary, this invention targets key molecules closely related to burns, such as VEGF-A and TGF-β1, and studies and analyzes the effects of ligustrazine on these molecular targets in a miniature pig model of second-degree burns, comparing and verifying the effects. It also delves into the regulatory effects of ligustrazine on VEGF-A and TGF-β1 in vivo and its molecular mechanism. Our results confirm that topical application of ligustrazine significantly promotes the healing of burn wounds. The mechanism is that ligustrazine (5g / kg) reduces the growth of bacteria in burn wounds by inhibiting the production and release of inflammatory mediators, thereby reducing the inflammatory response. Simultaneously, it upregulates the protein and gene expression levels of VEGF-A and TGF-β1, causing their expression peaks to occur earlier, thus shortening the time for eschar removal and hair regrowth in miniature pigs and improving the wound healing rate. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and to provide an application of scutellarin in the preparation of a drug for treating burns and scalds.

[0008] To address the problems of the prior art, the present invention provides the following technical solution:

[0009] Application of scald and burn preparations in the treatment of burns and scalds.

[0010] Preferably, the scalpelite is scalpelite monomer and / or scalpelite salt.

[0011] Preferably, the topical application of the 5g / kg of gentian violet reduces the growth of bacteria in burn wounds by inhibiting the production and release of inflammatory mediators, thereby alleviating the inflammatory response of the wound.

[0012] Preferably, the scutellarin emulsifier upregulates the protein and gene expression levels of VEGF-A and TGF-β1, causing their expression peaks to occur earlier.

[0013] Preferably, the styrax ethyl acetate can shorten the time for scab removal and hair regrowth in miniature pigs and improve the wound healing rate.

[0014] Preferably, the lamp scald has a significant therapeutic effect on burns.

[0015] Preferably, scutellarin is used to provide protection for burns and scalds for 7 days after administration.

[0016] Preferably, scutellarin has a protective effect 21 days after administration for the treatment of burns.

[0017] Furthermore, scutellarin can be formulated with excipients of appropriate dosage forms into one or more of the following: oral liquid, injection, tablet, pill, dispersant, capsule, soft capsule, drop pill, granule, solution, suspension, emulsion or microcapsule.

[0018] The beneficial effects of this invention are mainly reflected in:

[0019] Burns are not a single pathophysiological event, but a destructive injury causing structural and functional defects in multiple organ systems. Due to their complexity and the involvement of multiple organs, in vitro experiments cannot capture this complexity or resolve the pathophysiological issues. Over the past two decades, numerous animal models of burns (including mice and rats) have been developed to elucidate their pathophysiological progression. However, given the limitations and constraints of various burn models, the pathological progression in burn models differs significantly from the pathological changes in actual clinical burn patients. Since pig skin, including the density of body hair, epidermal thickness, subcutaneous fat layer, epidermal morphology and proliferative dynamics, as well as the fluid and metabolic changes after skin burns, are similar to those in humans, we conducted numerous experiments, strictly controlling the combustion time of burn fuel, to create a miniature pig model of second-degree burns using open flame. The pathological changes in this model are quite similar to the pathological development in actual clinical burn patients, allowing researchers to better understand these pathological changes for clinical translational applications. We established a second-degree miniature pig burn model and treated it with scutellarin and silver sulfadiazine. Western blot (WB) and real-time polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression levels of VEGF-A and TGF-β1 in the treated wound tissue at 0, 7, and 21 days after treatment. Bacterial cultures and counts were performed in each group on days 7 and 21 after treatment, and the levels of interleukin-2 (IL-2), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) in the wound tissue were measured using enzyme-linked immunosorbent assay (ELISA). HE staining and macroscopic monitoring of skin wound healing at different time points were also conducted.

[0020] The study results confirmed that topical application of scutellarin can significantly promote the healing of burn wounds. The mechanism is that scutellarin reduces the growth of bacteria in burn wounds by inhibiting the production and release of inflammatory mediators, thereby reducing the inflammatory response of the wound. At the same time, it is related to the upregulation of the protein and gene expression levels of VEGF-A and TGF-β1, causing their expression peak to occur earlier, thus shortening the time for scab removal and hair growth in miniature pigs and improving the wound healing rate.

[0021] Erigeron breviscapus extract (5g / kg) has a significant therapeutic effect on burns, even surpassing the efficacy of first-line clinical drugs such as 1% silver sulfadiazine (10g / kg). It can be used to prepare drugs for the prevention and / or treatment of burns. This invention fully explores the important applications of traditional medicine in disease prevention and treatment, providing a new approach for the clinical treatment of burns. Attached Figure Description

[0022] Figure 1 The chemical structural formula of scutellarin provided by this invention is shown below.

[0023] Figure 2 This is a schematic diagram of wound distribution in miniature pigs provided by the present invention.

[0024] Figure 3 Shows histological changes of skin after deep second-degree burn in miniature pigs provided by the present invention (HE, 100×).

[0025] Figure 4 Shows detection of protein and mRNA expression of VEGF-A and TGF-β1 by WB and RT-PCR provided by the present invention. Detailed Description of the Embodiments

[0026] The present invention is further described below by way of examples. It should be understood that these examples are for illustrative purposes only and do not limit the protection scope of the present invention in any way.

[0027] In the first embodiment, scutellarin is provided by Professor Zhang Renwei from Yunnan Institute of Materia Medica, with a purity of 98.8%. It is packaged in brown bottles and stored away from light in a dry place.

[0028] Example 1

[0029] Experiment of scutellarin for treatment of scald / burn.

[0030] 1. Establishment of second-degree burn model, administration, tissue collection and wound area measurement

[0031] Experimental animals and grouping

[0032] Twelve healthy adult Diannan breed miniature pigs, of either sex, numbered 1, 2, 3...12, aged 4 to 5 months, weighing 15 to 20 kg, were purchased from the Experimental Animal Center of Kunming Medical University (License No. for the use of standard experimental animals: SYXK (Dian) 2016-0004). The experimental feeding conditions are as follows: environment with room temperature of 25°C and humidity of 45%, 12 hours of light every day, standard experimental pig feed, fasting for 12 hours before operation. On both sides of the spine of each pig, 4 symmetric experimental areas (A, B, C, D) are delineated, each experimental area contains two wounds (e.g., A1, A2...). The 4 experimental areas are randomly divided into: blank control group (Control), pathological model group (Model), scutellarin group (Scutellarin), and silver sulfadiazine group (SSD). There are a total of 30 wounds in each experimental group, and 10 wounds are randomly distributed at each time point. The wound distribution of the miniature pigs is as Figure 2 shown.

[0033] After 12 hours of fasting before modeling, the hair on the back of the miniature pig is shaved off with an electric shaver, depilated with 25% Na2S (dissolved in alcohol), and anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg body weight). The miniature pig is placed in a prefabricated asbestos template with a rectangular opening (the area is about 5×8cm 2Exposing the pre-burned skin surface on the back while protecting the remaining skin from exposure. A mixed fuel (25ml gasoline, 60ml 95% alcohol, 60g rosin, 5ml glycerin, 5ml xylene) was evenly applied to the back of the miniature pig, 2ml per wound. After igniting for 20 seconds, the flame was quickly extinguished with a damp cloth, creating a second-degree burn wound in the miniature pig. The epidermis and superficial dermis were partially damaged, with significant inflammatory cell exudation and edema; skin appendages remained intact. The miniature pig presented typical clinical signs of second-degree burns.

[0034] Drug administration and material collection

[0035] Within 2 hours after burns, patients were divided into groups and administered medications. The model group received a 5% sodium carboxymethyl cellulose blank matrix, the scutellarin group received a uniform application of scutellarin preparation (5 g / kg) to the wound, and the silver sulfadiazine group received a uniform application of scutellarin preparation (10 g / kg). Treatment was administered once daily for 28 consecutive days. Skin samples were taken from the burn wounds at the same time points on days 0, 7, and 21. The wounds were sutured as soon as possible after sampling. On days 3, 7, and 21 post-treatment, wound secretions were collected from the entire wound using throat swabs and soaked in 10 ml of sterile water for injection, pending bacterial culture and counting.

[0036] 2. Observation indicators and detection methods

[0037] (1) HE-stained skin tissue was routinely dehydrated and embedded, and 4μm sections were prepared. After hematoxylin-eosin staining, the pathological changes of the skin tissue were observed under a microscope. Three cases of burns were randomly observed in each group, and five high-power fields were randomly selected from each section.

[0038] (2) Bacterial Count and ELISA: 15 μL of wound secretions were streaked evenly on 9 cm blood agar plates (China Blue Agar + Blood Agar) and MacConkey agar plates, and incubated at 37℃ for 24 h. Colonies were counted, and bacterial identification was performed using a bacterial identification instrument based on Gram staining microscopy and biochemical identification. Thawed tissue homogenate samples were analyzed according to the ELISA kit instructions to determine the levels of IL-2, IL-8, and TNF-α using the ELISA method, respectively, at ng·L⁻¹. -1 ,ng·L -1 pg·mL -1 express.

[0039] (3) Wound area measurement: The wound closure rate was used to represent the wound healing rate of piglets, and the wound area was tracked and measured. The actual values ​​were then converted into percentage values, with the wound size at the time of injury as 100%. A transparent chart was used every 7 days to monitor the gradual change in wound size. The wound area was measured using computer software (Media Cybernetics Inc, USA, IPP 6.0). The wound shrinkage rate was expressed as the percentage reduction in wound size, calculated as: Wound closure rate (%) = (A0 - An) / A0 × 100, where A0 and An are the wound areas at the initial wound (day 0) and day n, respectively.

[0040] (4) Western blot analysis: 2g of sample was taken from each wound and stored directly in a -80℃ freezer to detect the expression of VEGF-A and TGF-β1 proteins. Cells were repeatedly lysed with 400μL of RIPA protein lysis buffer and centrifuged at 12,000r / min for 10min at 4℃. The supernatant was collected. The protein concentration of each group of cells was determined by BCA method. Then, an appropriate amount of 4× protein loading buffer was added and the cells were boiled at 100℃ for 8min to denature them. A 10% separating gel was prepared, and the denatured proteins were subjected to SDS-PAGE electrophoresis (first electrophoresis at 80V for 30min, then electrophoresis at 120V for 1.5h). Wet transfer was performed (constant current: 250mA, time: 1.5h). After transfer, Anti-VEGF-A and TGF-β1 antibody (1:1000) were added. The membrane was washed the next day and horseradish enzyme-labeled goat anti-rabbit IgG (1:2000) was added. After 1 hour of incubation, ECL luminescence was observed for 3 minutes, and images were acquired using an Amersham Imager 600 system. The experiment was repeated three times, and the relative protein expression levels were determined by comparing the measured grayscale values ​​of each group with the corresponding internal control values ​​using ImageJ software.

[0041] (5) Real-time quantitative PCR (RT-PCR) detection: 2g of tissue was taken from each wound and immersed in RAN protection solution. The tissue was stored at 4-8℃ for 24h, and then transferred to -80℃ for storage. VEGF-A mRNA expression was detected. The procedure was strictly followed according to the instructions. Total RNA was extracted. cDNA synthesis: cDNA was synthesized using a 20μL total volume transcription reaction system prepared with Thermo's reverse transcription kit and stored at -80℃. The concentration and quality of the extracted total RNA were measured. Real-time quantitative PCR detection: A total volume of 20μL was prepared. The reaction system was prepared and the reaction was performed using a real-time quantitative PCR instrument. The reaction conditions were: pre-denaturation 95℃, 40s; denaturation 95℃, 15s; annealing and extension 60℃, 35s; 40 cycles. Each sample was tested three times. The relative mRNA expression level of each sample was calculated using the formula = 2. -△Ct△Ct = Ct value of target gene - Ct value of β-actin, and the obtained data were statistically analyzed. Primer design software Primer 5.0 was synthesized by Shuoqing Technology Co., Ltd.

[0042] TGF-β1(120bp):(Forward)5′-TTACAACAGTACCCGCGACC-3′;

[0043] (Reverse)5′-ATTTGGTTGCCCTTTCCAC-3′

[0044] VEGF-A(175bp):(Forward)5′-AGGCCAGCACATAGGAGAGA-3′,

[0045] (Reverse)5′-ACGCGAGTCTGTGTTTTTGC-3′

[0046] GAPDH(252bp):(Forward)5′-AGTTCCCACGGCACAGTCAAG-3′,

[0047] (Reverse)5′-TCTCATGGTTCACGCCCATC-3′.

[0048] 3. Results Analysis

[0049] 3.1 HE staining results

[0050] The skin tissue structure of the control group miniature pigs was clear, with basal cells arranged neatly in a cuboidal pattern. The epidermis and dermis were intact, without congestion or edema, and skin appendages were intact. 30 seconds after the burn, compared with the control group, the model group miniature pigs showed partial damage to the superficial structures of the epidermis and dermis, with extensive inflammatory cell exudation, significant edema, and intact skin appendages. The miniature pigs exhibited typical clinical signs of second-degree burns. Figure 3 .

[0051] 3.2 Bacterial count and ELISA results

[0052] (1) Bacterial culture and counting of wounds: Bacterial growth appeared in the control group on day 3 after treatment, while bacterial growth appeared in the scutellarin group and the silver sulfadiazine treatment group on day 7 after treatment. On days 7 and 21 after treatment, compared with the control group, the bacterial counts in the scutellarin and silver sulfadiazine treatment groups were significantly reduced (p<0.05), as shown in Table 1.

[0053] (2) Statistical analysis of pathogen distribution in wounds on days 7 and 21 after treatment in the model control group. Staphylococcus aureus and Pseudomonas aeruginosa had the highest detection rates in each group. Compared with day 21 after treatment, the detection rate of the above strains increased in the model control group, while it decreased in the scutellarin group and the silver sulfadiazine group (see Table 2).

[0054] (3) Comparison of IL-2, IL-8 and TNF-α levels in wound tissues of different groups. At 3, 7 and 21 days after treatment, compared with the model control group, the levels of IL-2, IL-8 and TNF-α in wound tissues treated with scutellaria baicalensis and silver sulfadiazine were significantly reduced (P<0.05), as shown in Table 3.

[0055] Table 1. Comparison of bacterial counts in wounds of different groups at 3, 7, and 21 days (n=10)

[0056]

[0057] Note: Compared with the control group 1) p < 0.05

[0058] Table 2. Distribution of pathogens on wounds at 7 and 21 days in each group.

[0059]

[0060]

[0061] Table 3 Comparison of IL-2, IL-8, and TNF-α levels in wound tissues of different groups (n=10, x±s)

[0062]

[0063] Note: Compared with the model control group 1) p < 0.05;

[0064] 3.3 The promoting effect of acetyl ether on a second-degree burn model

[0065] 3.3.1 Statistical analysis of healing rate, scab removal, and hair regrowth time

[0066] Compared with the silver sulfadiazine group and the scutellaria baicalensis group, the wound healing rate of the silver sulfadiazine group and the scutellaria baicalensis group were significantly improved, and the wound healing time was also significantly shortened, both showing statistically significant differences (P<0.05). The healing rates of the sulfadiazine group and the scutellaria baicalensis group were similar (P>0.05), as shown in Table 4.

[0067] Table 4 Comparison of healing rates among different groups at different time points (%)(n=10)

[0068]

[0069] Note: Compared with the model group, * P<0.05, ** P<0.01

[0070] 3.3.2 Statistical analysis of scab removal and hair regrowth time

[0071] Compared with the sulfadiazine silver group and the scutellaria baicalensis group, the model group showed shorter scab removal and hair growth time in both groups, with statistically significant differences (P<0.05). There was no statistically significant difference between the sulfadiazine silver group and the scutellaria baicalensis group (P>0.05), as shown in Table 5.

[0072] Table 5 Comparison of differences in scab removal and hair regrowth time among different groups (days)(n=30)

[0073]

[0074] Note: Compared with the model group, * P<0.05, ** P<0.01

[0075] 3.4 Western blot and RT-PCR detection of changes in VEGF-A and TGF-β1 protein and mRNA expression. Western blot and RT-PCR results showed that on the day of the burn experiment, all experimental groups showed a small amount of VEGF-A and TGF-β1 protein and mRNA expression, with no significant statistical differences between the groups (P>0.05). On the 7th day after the burn, the protein and mRNA expression of VEGF-A and TGF-β1 in the skin tissue of rats in the scutellarin and silver sulfadiazine groups increased sharply, reaching a peak value significantly higher than that in the pathological model group (P<0.001, P<0.001). There was no significant statistical difference between the scutellarin and silver sulfadiazine groups (P>0.05), suggesting that the therapeutic effect of these two drugs in shortening wound healing time may be related to the upregulation of growth factor expression. On day 21 post-burn, VEGF-A and TGF-β1 protein and mRNA levels in the model group reached their peak. Positive expression of protein and mRNA in the scutellarin and silver sulfadiazine groups continued to decline, approaching normal levels (P>0.05, P>0.05), while there was no significant statistical difference between the scutellarin and silver sulfadiazine groups (P>0.05). This invention suggests that the earlier peak expression of VEGF-A and TGF-β1 protein and mRNA after scutellarin intervention may indicate that "the expression of VEGF-A and TGF-β1 protein and mRNA is rapidly increased in the skin during burn trauma, and then begins to decline during the wound healing and recovery phase." This may be related to the fact that the drug-treated group can upregulate the total expression of VEGF-A and TGF-β1 protein and mRNA, thereby accelerating wound healing. Our research data confirms that, compared with the model group, the peak expression levels of VEGF-A and TGF-β1 in the ligustrazine group were earlier, which is consistent with the shortened eschar shedding time, hair regrowth time, and improved wound healing rate in piglets. Based on the effects of ligustrazine on VEGF-A and TGF-β1 in the skin tissue of burned piglets at different time points, it can be confirmed that the therapeutic effect of ligustrazine on burn wounds is achieved by upregulating the expression of VEGF-A and TGF-β1 protein and mRNA, thereby accelerating inflammation and cellular responses, promoting epithelialization of the wound, and ultimately promoting premature wound healing. (See [link to study]). Figure 4 .

[0076] The study included a blank control group (Control), a pathological model group (Model), a scutellarin group, and a silver sulfadiazine group (SSD); A, B, C, D, E, and F represent the protein and mRNA expression levels of VEGF-A and TGF-β1 in skin tissue at days 0, 7, and 21 of the burn experiment, respectively; compared with the normal control group. * P < 0.05, ** P < 0.01; compared with the model group #P < 0.05, ## P < 0.01,

[0077] Furthermore, the body has minimal adverse reactions to scald. This invention fully leverages the important applications of traditional medicine in disease prevention and treatment, providing a new approach for the clinical treatment of burns.

[0078] The conventional techniques described in the above embodiments are existing technologies known to those skilled in the art, and therefore will not be elaborated upon here. The above embodiments are merely illustrative of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also fall within the scope of the present invention, and the patent protection scope of the present invention should be defined by the claims.

Claims

1. The application of scutellarin preparations in the preparation of drugs for treating skin burns and scalds, characterized in that, The burns and scalds mentioned are flame burns and scalds, and the method of treating burns and scalds with the lamp ligustrazine is external application, which reduces the growth of bacteria in the burn and scald wound by inhibiting the production and release of inflammatory mediators and reducing the inflammatory response of the wound. The scalpelite is scalpelite monomer and / or scalpelite salt.

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