A hydrogel for treating atopic dermatitis and its application

Abstract

This invention provides a hydrogel for treating atopic dermatitis and its applications, relating to the field of biomedical technology. The hydrogel comprises SA, plasma water, L-G2, and / or FLA. Preliminary evaluation of the hydrogel was conducted through in vitro cytotoxicity and antibacterial performance tests, confirming its ability to inhibit Staphylococcus aureus and Escherichia coli. Simultaneously, the hydrogel exhibits low cytotoxicity, meeting the basic requirements for biomedical dressings. In animal experiments, by comparing skin lesions before and after treatment in AD mice, the hydrogel was observed to significantly improve skin lesions on the back of mice, demonstrating good anti-inflammatory effects. Pathological histological analysis showed that the hydrogel can reduce epidermal thickness, inhibit the increase in TNF-α levels induced by DNCB, and promote inflammation relief. Simultaneously, SA can provide a moist healing environment for skin wounds, helping to promote wound repair. Therefore, this hydrogel provides a new strategy for the treatment and management of AD and is expected to become a novel topical drug for AD treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a hydrogel for treating atopic dermatitis and its applications. Background Technology

[0002] Atopic dermatitis (AD), also known as atopic eczema or hereditary allergic dermatitis, is a chronic, relapsing, inflammatory skin disease that commonly affects children. In recent years, the prevalence of AD has been increasing globally, affecting 15-20% of children and 6-10% of adults. Of these patients, approximately 20% present with moderate to severe AD. AD patients of different ages often exhibit different skin lesion characteristics. Although there are differences in skin lesion characteristics among patients of different ages, itching is a common symptom of AD, and the severity of itching is correlated with the severity of the disease. Intense itching prompts scratching, further damaging the skin barrier, exacerbating the inflammatory response and itching, creating a vicious cycle of "itching-scratching-intensified itching." This persistent itching and scratching not only affects patients' daily lives and sleep but may also lead to psychological problems such as anxiety and depression, and even increase the risk of suicide. In addition to skin manifestations, Alzheimer's disease (AD) patients may also experience allergic diseases such as asthma and allergic rhinitis, as a complex chronic disease. Therefore, AD not only severely impacts patients' daily lives and work, reducing their quality of life, but also imposes a significant economic burden on their families and society.

[0003] The pathogenesis of Alzheimer's disease (AD) involves complex interactions of multiple factors, among which impaired skin barrier function is one of the main causes. Defects in the skin barrier create favorable conditions for the colonization and invasion of pathogenic microorganisms. Studies have shown that AD patients are more prone to skin flora imbalances, such as increased Staphylococcus aureus colonization. These bacteria can further negatively impact the integrity of the skin barrier and exacerbate the inflammatory response by secreting various virulence factors, such as superantigens, cytotoxins, proteases, and lipases. Therefore, alleviating skin inflammation and restoring normal skin barrier function are important means of treating AD lesions.

[0004] Currently, topical medications play a crucial role in the treatment of Alzheimer's disease (AD). Commonly used topical medications include corticosteroids (TCS), calcineurin inhibitors (TCI), and phosphodiesterase-4 (PDE-4) inhibitors. Among these, TCS is the most commonly used topical medication for AD. By acting on various inflammatory cells and factors such as macrophages and monocytes, it can rapidly relieve skin inflammation and reduce itching symptoms. However, long-term and excessive use can damage the skin barrier, leading to adverse reactions such as skin atrophy, telangiectasia, and hypopigmentation. TCI is the second most common treatment, including tacrolimus and pimecrolimus. Because long-term use does not damage the skin barrier, TCI is recommended for use on areas with thinner skin, such as the face, neck, and around the eyes, as well as special areas such as the perianal region and genitals, and for treating AD lesions in the subclinical and recovery phases. Crizoborone cream is currently the only PDE-4 inhibitor approved for clinical use and has been approved for the treatment of mild to moderate AD patients with symptoms of 3 months or more, demonstrating good efficacy in clinical applications. However, both TCI and PDE-4 inhibitors are prone to adverse reactions such as itching and pain at the application site during use. Furthermore, these topical medications cannot be used on broken skin lesions complicated by bacterial infection. Therefore, it is necessary to develop alternative therapies to address the shortcomings of existing treatment options. Summary of the Invention

[0005] The purpose of this invention is to address the limitations of existing topical medications for AD treatment, which also have technical problems such as inability to be used on broken skin and adverse reactions such as skin barrier damage, skin atrophy, telangiectasia, hypopigmentation, itching, and pain.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A hydrogel for treating atopic dermatitis includes sodium alginate (SA) and plasma water, and also includes L-G2 glycyrrhizin G2 and / or FLA glycyrrhizin isoflavone A.

[0008] Preferably, the hydrogel contains L-G2 and FLA, wherein the concentration of L-G2 is 50-150 μg / mL and the concentration of FLA is 150-260 μg / mL.

[0009] Preferably, the SA concentration in the hydrogel is 1-3%.

[0010] Preferably, the hydrogel is prepared as follows:

[0011] S1: L-G2 solution preparation: L-G2 powder is dissolved in deionized water to prepare L-G2 mother liquor;

[0012] S2: FLA solution preparation: Mix FLA powder with deionized water and stir thoroughly to form a uniformly dispersed suspension.

[0013] S3: Add an appropriate amount of deionized water to dilute the L-G2 mother liquor and FLA solution to achieve their respective working concentrations, then mix the two solutions. Finally, weigh SA according to the ratio and dissolve SA in the mixture of the two solutions.

[0014] Preferably, the concentration of the L-G2 mother liquor is 1 mg / mL, and the concentration of the FLA solution is 2 mg / mL.

[0015] This application also provides the use of glycyrrhizin G2 and / or glycyrrhizin isoflavone A in the preparation of medicaments for treating atopic dermatitis.

[0016] Compared with the prior art, this application has the following beneficial effects:

[0017] This application is based on the growing recognition of the potential value of herbal or natural product drugs in the treatment and management of Alzheimer's disease (AD), and the development research conducted in the context of exploring the use of various traditional Chinese medicine ingredients in AD treatment. Among them, licorice, as an important component of traditional Chinese medicine, relies on its rich active ingredients, including glycyrrhizin, glycyrrhizin flavonoids, and glycyrrhizin polysaccharides, to exert its anti-inflammatory effects. Glycyrrhizin, as one of the main active ingredients of licorice, can effectively inhibit the secretion of NO, TNF-α, and IL-6, thereby exerting its anti-inflammatory effects. Furthermore, it can further exert its anti-inflammatory effects by inhibiting PLA2 activity and COX-2 expression, and reducing the synthesis of PGE2 in the arachidonic acid metabolic pathway. Licoricesaponin G2 (L-G2), as a saponin, possesses the functions of the aforementioned saponins. Licorice isoflavone A (FLA), belonging to the flavonoid class of compounds, has been proven to have anti-inflammatory effects. In addition, studies have found that it can exert its antibacterial effect by inhibiting the formation of Staphylococcus aureus biofilm and the production of staphylococcal flavin.

[0018] This application constructs a novel and relatively safe hydrogel dressing and evaluates its therapeutic effects, cytotoxicity, and antibacterial properties through specific validation experiments, thereby providing a new approach for topical drug treatment of Alzheimer's disease (AD).

[0019] Specifically, this application conducted preliminary evaluations of the constructed novel composite hydrogel (SA+L-G2+LFA) through in vitro cytotoxicity and antibacterial performance tests, confirming its inhibitory effect on Staphylococcus aureus and Escherichia coli. Simultaneously, the hydrogel exhibited low cytotoxicity, meeting the basic requirements for biomedical dressings. In animal experiments, by comparing skin lesions before and after treatment in AD mice, it was observed that the SA+L-G2+LFA hydrogel significantly improved skin lesions on the back of mice. Pathological histological analysis showed that the hydrogel could reduce epidermal thickness, inhibit the increase in TNF-α levels induced by DNCB, and promote inflammation relief. Furthermore, sodium alginate hydrogel, as a carrier, can provide an ideal moist healing environment for skin wounds, helping to promote skin repair. Therefore, this hydrogel provides a new strategy for the treatment and management of AD and can become a novel topical drug for AD treatment. Attached Figure Description

[0020] Figure 1 This is an experiment on the viscosity of sodium alginate hydrogels of different concentrations in Example 1 of the present invention;

[0021] Figure 2 This is a SEM image of the SA+L-G2+LFA hydrogel in Example 2 of the present invention;

[0022] Figure 3 This is a comparison of cell viability after co-incubation of L929 cells and 293T cells with different concentrations of SA hydrogel in Example 3 of the present invention;

[0023] Figure 4 The images show the fluorescence staining of live / dead cells after co-incubation with different concentrations of SA hydrogel in one embodiment of the present invention: (a) 293T cells; (b) L292 cells.

[0024] Figure 5 This invention provides a comparative analysis of the antibacterial properties of different hydrogels in one embodiment, including (a) the results of the inhibition zone experiments of SA hydrogel, SA+L-G2 hydrogel, SA+LFA hydrogel, and SA+L-G2+LFA hydrogel; and (b) a quantitative analysis diagram of the inhibition zone size. For the analysis of significant differences, each experimental group was compared with the SA+L-G2+LFA hydrogel group. * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0025] Figure 6 This is a comparison of skin lesions in mice before and after treatment in one embodiment of the present invention;

[0026] Figure 7For comparison of SCORAD scores of dorsal skin lesions in mice in one embodiment of the present invention: (a) Day 0 of treatment; (b) Day 7 of treatment; (c) Day 14 of treatment. For statistical significance analysis, * represents p<0.05, ** represents p<0.01;

[0027] Figure 8 This is a comparison of H&E staining results of mouse back skin tissue in one embodiment of the present invention;

[0028] Figure 9 This is a comparison image of MASSON staining results of mouse back skin tissue in one embodiment of the present invention;

[0029] Figure 10 To illustrate the effect of SA+L-G2+LFA hydrogel on IL-10 expression levels in skin lesions of AD mice in one embodiment of the present invention, (a) immunofluorescence image; (b) quantitative analysis image. Significant differences were analyzed: Untreated group vs. GC group, Untreated group vs. SA+L-G2+LFA hydrogel group. *** represents p<0.001.

[0030] Figure 11 This invention presents a comparative study of the effects of SA+L-G2+LFA hydrogel on TNF-α expression levels in skin lesions of AD mice, as shown in (a) immunofluorescence image and (b) quantitative analysis image. For significant differences, the analysis compares the effects of untreated group vs. GC group and untreated group vs. SA+L-G2+LFA hydrogel group. * represents p < 0.05, ** represents p < 0.01. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments.

[0032] A hydrogel for treating atopic dermatitis includes sodium alginate (SA) and plasma water, and also includes glycyrrhizin G2 (L-G2) and / or glycyrrhizin isoflavone A (FLA).

[0033] Preferably, the hydrogel contains glycyrrhizin G2 and glycyrrhizin isoflavone A, wherein the concentration of glycyrrhizin G2 is 50-150 μg / mL, the concentration of glycyrrhizin isoflavone A is 150-260 μg / mL, and the concentration of SA is 1-3%, preferably 3%.

[0034] This application also provides a method for preparing the hydrogel described above, the steps of which are as follows:

[0035] S1: L-G2 solution preparation: Dissolve L-G2 powder in deionized water to prepare a stock solution with a concentration of 1 mg / mL.

[0036] S2: FLA solution preparation: Mix FLA powder with deionized water and stir thoroughly to form a uniformly dispersed suspension (2 mg / mL stock solution).

[0037] S3: Mixed solution with added SA: Take both stock solutions into centrifuge tubes and dilute with appropriate amounts of deionized water to bring L-G2 and FLA to their respective working concentrations (100 μg / mL and 200 μg / mL, respectively); then weigh SA according to the concentration ratio and dissolve it in the above diluent.

[0038] S4: Storage method: Store at 4℃, sealed, away from light and prevent moisture evaporation. Shelf life is 2 months.

[0039] In addition, this application also provides the use of glycyrrhizin G2 and / or glycyrrhizin isoflavone A in the preparation of medicaments for treating atopic dermatitis.

[0040] The above content will be explained in conjunction with specific experiments:

[0041] Example 1: Preparation of SA+L-G2+LFA hydrogel

[0042] First, select the appropriate SA concentration. Prepare three centrifuge tubes labeled with SA concentrations (1%, 2%, and 3%) and dates. Weigh a measured amount of SA powder into each tube and add deionized water in the correct proportions to fully dissolve the SA powder. Transfer equal volumes of SA hydrogels of different concentrations into small sample bottles, tilt the bottles, and observe the viscosity of the hydrogels. Use a pipette to take 1 mL of hydrogel from each sample and place it on the lab bench. Use a cotton swab to pick up the hydrogel and pull it upwards, observing the viscosity of the hydrogels at different concentrations.

[0043] After determining the SA concentration, L-G2 powder was dissolved in deionized water to prepare a stock solution with a concentration of 1 mg / mL. Simultaneously, FLA powder was mixed with deionized water and stirred thoroughly to form a uniformly dispersed suspension (2 mg / mL stock solution). Next, both stock solutions were placed in centrifuge tubes and diluted with appropriate amounts of deionized water to achieve their respective working concentrations (100 μg / mL and 200 μg / mL, respectively). A certain amount of SA was weighed out according to the ratio and dissolved in the above diluents.

[0044] Please see Figure 1 , Figure 1 The viscous properties of three different concentrations of SA hydrogels were demonstrated. As shown in the figure, the swab was pulled upwards the greatest distance when the SA concentration was 3%, indicating that the hydrogel exhibited the best viscosity at this concentration. Based on these results, future research will use a 3% SA concentration as a base to prepare SA+L-G2+LFA hydrogels.

[0045] Example 2: Characterization of the structure and physical properties of SA+L-G2+LFA hydrogel

[0046] After the prepared SA+L-G2+LFA hydrogel was thoroughly freeze-dried, its cross-sectional microstructure was observed and images were acquired using a scanning electron microscope.

[0047] To observe the microstructure of the SA+L-G2+LFA hydrogel dressing, this application uses SEM to observe the cross-section of the lyophilized hydrogel. Under the microscope, the SA+L-G2+LFA hydrogel exhibits a porous structure composed of interconnected pores of varying sizes, resembling a honeycomb. Figure 2 This loose and porous microstructure not only endows the hydrogel with excellent structural stability and mechanical strength, but also provides significant advantages for its clinical application in dressings. Its porosity facilitates drug absorption and release, and also efficiently absorbs wound exudate, creating a more ideal microenvironment for wound healing. These properties indicate that the microstructure design of the SA+L-G2+LFA hydrogel dressing has potential value in promoting wound repair.

[0048] Example 3: In vitro cytotoxicity experiment:

[0049] The in vitro cytotoxicity of the hydrogels was evaluated using mouse epithelial-like fibroblasts (L929 cells) and human embryonic kidney cells (293T cells). Both cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DEME medium.

[0050] (1) Cell resuscitation: Remove the frozen cells from the liquid nitrogen tank and thaw them in a 37°C water bath. After spraying with alcohol, proceed with subsequent operations on the operating table. Transfer the cell suspension to a centrifuge tube containing 5 mL of culture medium and centrifuge (1000 rpm / 3 min). Prepare cell culture flasks and label them with the date, cell name, and user's name. After centrifugation, discard the supernatant in the tube, add 1 mL of culture medium to resuspend the cells, mix well by pipetting, and transfer the cell-containing culture medium to a culture flask. Add an appropriate amount of culture medium. Place the culture flask in a cell culture incubator containing 5% carbon dioxide and incubate at 37°C.

[0051] (2) Cell Seeding: After observing cell adhesion under a microscope, aspirate the culture medium and wash three times with sterile PBS to remove dead cells and metabolic impurities. Add an appropriate amount of trypsin to the culture flask, place it in an incubator for 2 minutes of digestion, and observe under a microscope. The cells should appear round and free. At this point, add 2 mL of culture medium to stop the digestion. Use a pipette to repeatedly pipette the cells to ensure they detach fully from the flask wall. Transfer the cell suspension from the culture flask to a centrifuge tube and centrifuge (1000 rpm / 3 min). Discard the supernatant and take an appropriate amount of cell suspension. Dilute with culture medium and add approximately 100 μL to each well of a 96-well plate, resulting in a cell density of approximately 5000 cells / well. Use four wells per group, and add PBS solution to the surrounding wells to reduce the impact of evaporation. Place the plate in a cell culture incubator and incubate for 12 hours until the cells adhere.

[0052] (3) Drug treatment and incubation: The prepared SA hydrogel was dried and crushed to prepare suspensions of different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL) and sterilized with ultraviolet light. The original culture medium was aspirated, and the experimental groups were successively added with culture medium containing different concentrations of hydrogel, while the control group was added with normal culture medium. In addition, a blank group was set up with normal culture medium without cells. Incubation continued for 24 hours.

[0053] (4) CCK-8 incubation: Add 10 μL of CCK-8 solution to each well under dark conditions, and continue incubation in an incubator for 2 hours. Then, measure the absorbance (OD) of the cells at 450 nm using a microplate reader, and calculate the cell viability based on the OD.

[0054] Cell viability (%) = (OD hydrogel - OD blank group) / (OD control group - OD blank group) × 100%

[0055] Experimental results show that ( Figure 3 As the SA concentration increased, the in vitro survival rates of L292 cells and 293T cells decreased slightly, but even at an SA concentration of 500 μg / mL, the survival rates of both cell types remained above 80%. When the SA concentration was 50 μg / mL, the survival rates of L929 cells and 293T cells were not significantly different from the control group (without hydrogel), and the survival rates were close to 100%.

[0056] (5) Fluorescent staining: 293T and L929 cell suspensions were seeded into 12-well plates, 1 mL per well, at a cell density of approximately 5000 cells / well. Cells were incubated for 12 hours until adherence. The supernatant was aspirated and the cells were washed with PBS. 1 mL of culture medium containing different concentrations of hydrogel (50, 100, 200, 300, 400, 500 μg / mL) was added to each well sequentially. Incubation was continued for 24 hours, followed by digestion and centrifugation. Cells were then diluted with 1×Assay Buffer to a density of 1×10⁻⁶. 5 -1×10 6 / mL of cell suspension. Take 200μL of each cell suspension and put it into a 12-well plate, and add 100μL of staining working solution (Calcein-AM:PI:1×AssayBuffer=1:3:1000). Incubate at 37℃ for 15 min and then observe under a fluorescence microscope.

[0057] Fluorescent staining results as follows Figure 4 As shown in (a, b), after one day of co-culture, all concentration groups maintained high viability, with a large number of live cells (green fluorescence signal) visible, while the number of dead cells (red fluorescence) was significantly less than that of live cells. This further confirms that even at higher concentrations, SA hydrogel still exhibits low biotoxicity and does not significantly inhibit cell growth.

[0058] In summary, SA exhibits good safety profiles, which lays the foundation for the application of SA+L-G2+FLA hydrogel as a wound dressing.

[0059] Example 4: In vitro antibacterial performance test

[0060] Using Escherichia coli and Staphylococcus aureus as bacterial models, the antibacterial properties of the hydrogel were evaluated using the inhibition zone method.

[0061] (1) Weigh out the required proportions of tryptone, sucrose, sodium chloride, and yeast powder, place them in a beaker, and add 400 mL of deionized water. Place the beaker on a magnetic stirrer to fully dissolve and mix the solutes. Divide the mixed liquid culture medium into two 500 mL Erlenmeyer flasks. Cover the mouth of the flask with aluminum foil and then transfer the Erlenmeyer flasks to an autoclave for sterilization (121 °C, 30 min). After sterilization, transfer the Erlenmeyer flasks to a laminar flow hood and allow them to cool to room temperature.

[0062] (2) Take the required bacterial suspension from the -80℃ freezer, thaw it at room temperature, and add 100 μL to the cooled liquid culture medium. Incubate the culture medium containing the bacterial suspension at 37℃ and 120 rpm for 12 h.

[0063] (3) Weigh agar, tryptone, sucrose, sodium chloride, and yeast powder according to the required proportions, add 100 mL of deionized water to dissolve them completely, and then sterilize in an autoclave for 30 min. When the culture medium cools to about 50°C, take 1 mL of the bacterial suspension and mix it evenly. Pour the agar containing the bacterial suspension into a petri dish, and after solidification, you will obtain the bacterial agar medium. Use a punch to make agar-free well with a diameter of about 7 mm in the center of the agar plate and add SA hydrogel, SA+L-G2 hydrogel, SA+LFA hydrogel, and SA+L-G2+LFA hydrogel respectively. Transfer the petri dish to a constant temperature incubator at 37°C and incubate for 24 h. The antibacterial activity of each hydrogel is evaluated by measuring the diameter of the inhibition zone.

[0064] The results are as follows Figure 5 As shown in (a, b), for *Escherichia coli*, the SA hydrogel alone did not exhibit significant antibacterial activity, while both the SA+L-G2 and SA+LFA hydrogels showed some antibacterial effect. The inhibition zones formed by the two were similar in size, with radii of 1.2 cm and 1.3 cm, respectively. In contrast, the inhibition zone formed by the SA+L-G2+LFA hydrogel was more significant, reaching a radius of 2.1 cm. A similar trend was observed in the *Staphylococcus aureus* group; compared to hydrogels loaded with L-G2 or LFA alone, the SA+L-G2+LFA hydrogel showed superior antibacterial effect, with an inhibition zone radius reaching 1.5 cm.

[0065] Example 5: Establishment and drug administration of an AD mouse model

[0066] Fifteen BALB / c mice were selected. Three days before the experiment, the hair on the backs of the mice was shaved with an electric razor, and Veet hair removal cream was applied. After 5 minutes, the area was wiped clean with purified water, creating a 2.5cm × 2.5cm hairless area on the backs of the mice. Previous studies have demonstrated that repeated stimulation of mouse skin with DNCB can successfully induce AD-like lesions. Therefore, this experiment used DNCB to induce an AD mouse model.

[0067] In the experiment, acetone and olive oil were mixed in a 4:1 ratio to prepare a base solution, which was then used to dilute DNCB, resulting in DNCB solutions with concentrations of 1.5% and 0.15%. During the first week of the experiment, mice were sensitized by applying 100 μL of the 1.5% DNCB solution to the skin on their backs daily.

[0068] Mice that successfully modeled the allergen were randomly assigned to three groups: the AD group (Untreated group), the glucocorticoid treatment group (GC group), and the SA+L-G2+LFA hydrogel group. Starting from the second week of the experiment, the GC group and the SA+L-G2+LFA hydrogel group received glucocorticoids and therapeutic hydrogels, respectively, applied to the skin lesions. The Untreated group received no special treatment for comparison. Treatment was administered twice daily for two weeks. During the treatment period, each group of mice received 100 μL of 0.15% DNCB solution applied to their back skin every three days to simulate continuous allergen exposure. Changes in the skin lesions on the back of the mice were photographed every two days.

[0069] Please see Figure 6 , Figure 6 Representative images of skin lesions in mice before and after treatment are shown. In this application, day 7 of drug administration is defined as the start of treatment (day 0 of treatment), at which time obvious erythema, papules, exudation and crust-like changes appeared on the back of DNCB-sensitized mice.

[0070] On day 7 of treatment, the skin lesions on the backs of mice in the Untreated group showed no significant improvement, with persistent significant erythema and papules, accompanied by dry skin and extensive desquamation. Some mice exhibited noticeable exudation at the lesions, and after partial scab removal, erythematous erosions were visible, indicating that the inflammatory response was ongoing. In contrast, the skin lesions on the backs of mice in the GC group showed significant improvement, with a reduction in the area of ​​erythema and papules compared to before treatment. After some scabs fell off, relatively normal new skin was visible, but dry skin and desquamation remained at this stage. The skin lesions of mice treated with SA+L-G2+LFA hydrogel showed similar improvement to the GC group, but with more significant improvement in dry skin and desquamation. The skin color on their backs was close to that of normal mice, and a small amount of new hair growth was visible.

[0071] On day 14 of treatment, although the skin lesions in the Untreated group mice showed improvement compared to before, dry skin was still quite noticeable, accompanied by localized erythema and desquamation, and some severely affected areas were still covered with scabs. Compared to the Untreated group, the papules on the backs of the GC group mice had largely subsided, and the skin color was similar to that of normal mice, with only a few areas showing slight erythema, and new hair growth was also visible on the backs. At this time, the skin on the backs of the mice in the SA+L-G2+LFA hydrogel group had basically returned to normal, and a large amount of new hair growth was visible on the backs.

[0072] Example 6: SCORAD Score

[0073] On day 7 of modeling and on days 7 and 14 of treatment, the SCORAD score was used to assess the skin lesions on the backs of the mice. The specific scoring criteria are as follows:

[0074] (1) First, estimate the proportion of the mouse skin lesion area to the bare skin and divide it into 4 grades: Grade 1 (skin lesion area < 25%), Grade 2 (25% ≤ skin lesion area < 50%), Grade 3 (50 ≤ skin lesion area < 75%) and Grade 4 (skin lesion area ≥ 75%).

[0075] (2) Next, the erythema, exudation or crusting, peeling, lichenification and edema on the bare skin of mice were scored one by one. The scoring criteria were divided into 1-4 grades according to the area of ​​skin lesions, and each symptom was assigned 1-4 points.

[0076] With increasing DNCB sensitization frequency, the skin lesions on the backs of mice significantly worsened. By day 7 of treatment (day 0), all groups of mice exhibited obvious AD-like skin lesions on their backs. At this point, the SCORAD score of the skin lesions on the backs of all groups of mice significantly increased, reaching 15 points, and there was no statistically significant difference between groups. Figure 7 a) After treatment intervention, the severity of skin lesions on the backs of mice showed a time-dependent change, as shown in the following results. Figure 7 As shown in (b, c). On day 7 of treatment, the GC group score decreased to 6 points, while the SA+L-G2+LFA hydrogel group score decreased to 3.5 points, showing a statistically significant difference compared to the Untreated group (p<0.01). By day 14 of treatment, this improvement trend was more pronounced, with both the GC group and the SA+L-G2+LFA hydrogel group scores lower than the Untreated group. The SA+L-G2+LFA hydrogel group showed a better therapeutic effect than the GC group. The SCORAD scores of the mice in each group at the above different time points were consistent with the skin lesion changes in Example 4.

[0077] Example 7: Preparation of frozen slices:

[0078] On day 14 of treatment, the skin inflammation symptoms on the backs of mice in the hydrogel group had largely disappeared. At this point, all experimental mice in each group were euthanized and processed. Skin samples of approximately 2cm × 2cm size were cut from the backs of mice at the effective drug administration sites and placed in centrifuge tubes, where 4% paraformaldehyde was added for fixation for 24 hours. Each centrifuge tube was labeled with the mouse's number, the time of drug administration, and the drug used in the treatment. After fixation, the skin tissue was removed, washed with PBS solution, and then sequentially placed in 15% and 30% sucrose solutions for 24 hours for gradient dehydration.

[0079] Start the cryostat and set the section chamber temperature to -20°C. Remove the mouse skin sample that has sunk to the bottom of the 30% sucrose solution, cut it to an appropriate size and shape, and blot off excess sucrose solution with absorbent filter paper to ensure the skin cross-section is parallel to the embedding cassette. Add an appropriate amount of OCT embedding medium to completely cover the skin tissue and place the embedding cassette in the quick-freezing area of ​​the section chamber. After the OCT embedding medium has solidified, fix the embedding cassette on the sample holder and adjust the specimen position. Attach the blade to the blade holder, set the section thickness to 50-80 μm, and trim off any excess solid OCT embedding medium. When the skin tissue is exposed, install the anti-roll plate and adjust its position to ensure that subsequent cut skin tissue sections do not curl. Simultaneously, adjust the section thickness to 10 μm and proceed with subsequent sectioning. Mark the mouse number and experimental date on the slide with a pencil beforehand for section collection. After cutting the appropriate skin tissue, remove the slide from the section chamber and bring it close to the skin tissue to ensure complete attachment. After all skin sections are prepared, the slides are transferred to the slide box and stored in a refrigerator at -20°C.

[0080] Example 8: H&E staining

[0081] First, remove the pre-prepared frozen sections and allow them to air dry at room temperature. Then, place the sections in a humidified chamber, add cold acetone to the surface, add a small amount of water to the bottom of the chamber to prevent acetone evaporation, and place it in a 4°C freezer for 10 minutes to fix the sections. After removing the sections, wash them twice with PBS buffer, 1 minute each time. Immerse the slides in hematoxylin staining solution for 5 minutes, then gently rinse with water for 5-10 seconds. Immerse the slides in 1% hydrochloric acid ethanol for 2 seconds, then rinse again with water. Blot away excess water with filter paper and observe under a microscope to see if the cell nuclei are blue-purple. Next, add 1% ammonia solution to the surface of the sections for 5-10 seconds, rinse with water and wipe away excess water; under this point, the cell nuclei should appear blue. Immerse the slides in eosin staining solution for 30-60 seconds, then rinse away excess stain with water. The stained slides were sequentially dehydrated and cleared using ethanol solutions of different concentrations (75%, 85%, 95%, and 100%) and xylene solutions. After ensuring no xylene solution residue remained on the slides, they were mounted with neutral resin. Finally, images were acquired and analyzed under a microscope.

[0082] HE staining results showed that ( Figure 8In the untreated group, mouse skin samples showed a significant increase in epidermal thickness, along with a substantial reduction in the number of hair follicles in the dermis, making normal hair follicle structures almost indistinguishable. In contrast, mice treated with TCS and SA+L-G2+LFA hydrogels exhibited reduced epidermal thickness and varying degrees of hair follicle recovery. The SA+L-G2+LFA hydrogel treatment group showed the most significant recovery in hair follicle count, indicating effective inflammation relief and a near-normal skin tissue condition.

[0083] Example 9: MASSON staining

[0084] The fixation steps for the sections are as described above. After fixation, nuclear staining is performed first. Weigert iron hematoxylin stain is applied to the surface of the section and stained for 5-10 minutes, followed by rinsing with water to remove excess stain and loose dye. Excess water is then absorbed with filter paper. Next, the slide is immersed in a 1% hydrochloric acid-ethanol solution for differentiation, and rinsed with running water after 5-10 seconds. Cytoplasmic staining is then performed, with a prepared Ponceau S-acid-fuchsin solution applied to the surface of the section and stained for 5-10 minutes. After rapid rinsing with water, the slide is treated with a phosphomolybdic acid aqueous solution for 3 minutes. Finally, collagen fiber staining is performed, counterstaining with aniline blue for 5 minutes, followed by treatment with a 1% glacial acetic acid solution for 1 minute. The sections are then sequentially immersed in ethanol and xylene solutions of different concentrations for dehydration and clearing. Images are acquired after mounting.

[0085] MASSON staining results showed ( Figure 9 In the skin samples of mice in the untreated group, the collagen fibers were arranged in a disordered manner, with some collagen fibers showing signs of breakage and significantly increased gaps between fibers. The collagen fibers in the GC group showed similar characteristics to those in the untreated group. However, in the SA+L-G2+LFA hydrogel group, the collagen fibers were arranged in an orderly and dense manner. Furthermore, compared to the untreated and GC groups, the recovery of hair follicle count was most significant in the SA+L-G2+LFA hydrogel group, a result consistent with the HE staining results mentioned above.

[0086] Example 10: Immunofluorescence

[0087] After fixation, the sections were placed in a 1.2% hydrogen peroxide solution and incubated in the dark for 30 minutes to thoroughly remove non-specific staining. They were then washed three times with PBS buffer, 10 minutes each time. After wiping away excess water, Triton-100 was diluted to 0.3% with PBS solution, and an appropriate amount was added to cover the tissue. The tissue was then treated at room temperature for 30 minutes. The sections were rinsed again with PBS solution to remove excess Triton-100. Once the sections were slightly dry, a circle was drawn around the tissue using a histochemical pen. The required amount of bovine serum albumin powder was weighed and dissolved in an appropriate amount of deionized water to prepare a 3% BSA solution. The prepared BSA solution was added dropwise to the circled area, ensuring even coverage of the tissue, and treated for 30 minutes. After the primary antibodies (IL-10 Rabbit Polyclonal Antibody and TNF-α Rabbit Polyclonal Antibody) thawed at room temperature, 1 μL of each was pipetteed into a centrifuge tube, and antibody dilution buffer (PBS solution containing 1% BSA) was added to dilute to the working concentration at a ratio of 1:500. The slides were placed in a humidified chamber and the prepared primary antibody solution was added, then incubated overnight at 4°C. After primary antibody incubation, the slides were washed three times with PBS solution. After wiping away excess water, FITC-IgG fluorescent antibody (diluted 1:300 in PBS) was added, and the slides were incubated at room temperature in the dark for 2 hours. Finally, excess antibody was washed away with PBS solution, and the slides were mounted with buffered glycerol. Images were then acquired under a confocal fluorescence microscope.

[0088] In the immunofluorescence staining pattern ( Figure 10 a) Significant green fluorescence signal was observed in the skin samples of the untreated group, indicating a higher expression level of IL-10. In contrast, the green fluorescence signal was significantly weakened in the groups treated with TCS and SA+L-G2+LFA hydrogels. Quantitative analysis results of the mean fluorescence intensity of IL-10 ( Figure 10 (b) This also indicates that the expression level of IL-10 in the skin lesions of mice in the GC group and the SA+L-G2+LFA hydrogel group was significantly lower than that in the untreated group (p<0.001). IL-10 can be secreted by various cells such as Th2 cells, mast cells, and M2 macrophages, and is an important anti-inflammatory factor. It limits excessive immune responses by inhibiting the production of pro-inflammatory factors and downregulating the expression of MHC-II and co-stimulatory molecules. The expression level of IL-10 was the highest in the skin samples of the untreated group, which may reflect a compensatory anti-inflammatory response. The decrease in IL-10 expression level after treatment with TCS and SA+L-G2+LFA hydrogels may be closely related to the reduction of inflammatory response at the skin lesion site, a result consistent with the improvement trend of skin lesions on the back of mice.

[0089] In addition, this application also detected the expression level of TNF-α in the dorsal skin lesions of AD mice. Immunofluorescence staining patterns and quantitative analysis of mean fluorescence intensity showed ( Figure 11 In the untreated group, TNF-α expression was high, with the most significant green fluorescence signal. While the fluorescence signal in the GC group was somewhat weakened (p < 0.05), it still maintained high expression. The green fluorescence signal in the SA+L-G2+LFA hydrogel group was significantly weakened. TNF-α, as an important pro-inflammatory factor, has an expression level closely related to the degree of inflammation. The high TNF-α expression in the untreated group indicates an active inflammatory response. After treatment with TCS and SA+L-G2+LFA hydrogel, TNF-α expression levels decreased, indicating a weakened inflammatory response. Furthermore, the SA+L-G2+LFA hydrogel treatment was more effective than the GC group, with the lowest TNF-α expression level in its skin samples. These results suggest that SA+L-G2+LFA hydrogel may effectively inhibit the inflammatory response and exert an anti-inflammatory effect by downregulating TNF-α expression.

[0090] All data in this application are expressed as mean ± standard deviation. GraphPadPrism 8 statistical software was used for calculation and analysis, and One-way ANOVA was used for significance analysis. Statistical significance was set as *p<0.05, **p<0.01, and ***p<0.001.

[0091] The experimental reagents and equipment used in this application are shown below:

[0092] 1. Experimental reagents

[0093]

[0094]

[0095] 2. Experimental apparatus:

[0096]

[0097] 3. Laboratory animals:

[0098] SPF (Specific Pathogen Free) grade male BALB / c mice (4-6 weeks old) were purchased from the Experimental Animal Center of Nantong University. During the experiment, the mice were housed at a temperature of 20-25℃ and a humidity of 50-70%, with a 12-hour on / 12-hour off lighting cycle. All mice had free access to food and water. Mice were acclimatized to the environment for one week prior to the experiment. All experimental animals used in this experiment were approved by the ethics committee (P20250225-065).

[0099] In summary, this application first evaluated the safety of the SA+L-G2+LFA hydrogel and its inhibitory effect on Staphylococcus aureus and Escherichia coli through in vitro cell experiments. Simultaneously, this application further validated the therapeutic effect of the hydrogel using a DNCB-induced AD mouse model. In addition to observing and recording changes in AD skin lesions, this application also confirmed its therapeutic effect from a histopathological perspective by detecting the decrease in TNF-α expression levels, IL-10 reduction, and epidermal thickness in mouse skin tissue. This application focuses on two active substances derived from licorice: L-G2 (a saponin compound) and LFA (a flavonoid compound). The antibacterial experimental results of this application are consistent with some conclusions of the above experiments, demonstrating that both L-G2 and LFA have good antibacterial activity and anti-inflammatory effects, significantly inhibiting the growth of Staphylococcus aureus and Escherichia coli. The effect is even more significant when these two compounds are used in combination. Furthermore, sodium alginate hydrogel, as a carrier, can provide an ideal moist healing environment for skin wounds, helping to promote skin repair. Based on this, the hydrogel provided in this application for the treatment of atopic dermatitis offers a new strategy for the treatment and management of AD, and is expected to become a novel topical drug for the treatment of AD.

Claims

1. A hydrogel for the treatment of atopic dermatitis, characterized in that: It includes sodium alginate (SA) and plasma water, as well as L-G2 glycyrrhizin G2 and FLA glycyrrhizin isoflavone A; wherein the concentration of sodium alginate (SA) is 3%, the concentration of L-G2 glycyrrhizin G2 is 100 μg / mL, and the concentration of FLA glycyrrhizin isoflavone A is 200 μg / mL.

2. The hydrogel for treating atopic dermatitis according to claim 1, wherein: The hydrogel preparation method is as follows: S1: L-G2 solution preparation: L-G2 powder is dissolved in deionized water to prepare L-G2 mother liquor; S2: FLA solution preparation: Mix FLA powder with deionized water and stir thoroughly to form a uniformly dispersed suspension. S3: Dilute the L-G2 mother liquor and FLA solution with an appropriate amount of deionized water to achieve working concentrations of 100 μg / mL and 200 μg / mL, respectively. Then, mix the two solutions together. Weigh SA at a concentration of 3% and dissolve SA in the mixture of the two solutions.

3. The hydrogel for treating atopic dermatitis according to claim 2, characterized in that: The concentration of the L-G2 mother liquor is 1 mg / mL, and the concentration of the FLA solution is 2 mg / mL.

4. The application of glycyrrhizin G2 and glycyrrhizin isoflavone A in the preparation of drugs for treating atopic dermatitis, characterized in that, The concentration of glycyrrhizin G2 is 100 μg / mL, the concentration of glycyrrhizin isoflavone A is 200 μg / mL, and the drug carrier is a 3% sodium alginate SA hydrogel.

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