A pharmaceutical preparation for treating atopic dermatitis and a method for preparing the same

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical preparation for treating atopic dermatitis and its preparation method. The pharmaceutical preparation comprises the following raw materials in parts by weight: 10-15 parts recombinant CFL1 protein, 1.5-3 parts *Hymenochaete scabra* polysaccharide, 18-22 parts sodium alginate, 10-16 parts poloxamer 407, 1-2 parts ascorbic acid, and 34-45 parts water. Experimental results show that applying the pharmaceutical preparation prepared according to this invention can significantly alleviate symptoms such as skin inflammation, edema, and scaling in animals, reduce inflammatory responses, and effectively relieve itching behavior in mice. It has significant implications for the treatment of atopic dermatitis in mice and has promising market application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical preparation for treating atopic dermatitis and its preparation method. Background Technology

[0002] Atopic dermatitis (AD) is a common, chronic, relapsing, inflammatory skin disease characterized by intense itching, eczematous lesions, and impaired skin barrier function. The condition is persistent and recurring, significantly impacting children's growth and development, physical and mental health, and quality of life. Current clinical treatment primarily relies on anti-inflammatory drugs such as corticosteroids and calcineurin inhibitors. While these can alleviate symptoms to some extent, long-term use can lead to serious adverse reactions such as skin atrophy, telangiectasia, immunosuppression, and hormone dependence. Therefore, developing novel, safe, and effective AD treatments is of significant clinical importance and a practical necessity.

[0003] CFL1 is an actin-binding protein primarily involved in cytoskeleton remodeling. Previous studies have shown that CFL1 is significantly overexpressed in the serum and skin lesions of AD patients, and its abnormal expression is closely related to T cell activation, the release of inflammatory cytokines, and skin barrier disruption. However, there are no reports of using recombinant CFL1 protein directly as an exogenous therapeutic agent for AD treatment. Furthermore, maintaining the bioactivity of recombinant proteins and promoting their transdermal penetration in topical delivery systems remains a technical bottleneck restricting the local application of protein drugs.

[0004] Given the shortcomings of the existing technologies, there is an urgent need to develop a novel topical drug formulation that repairs the skin barrier function, has high safety, and good patient compliance, in order to meet the treatment needs of long-term AD management. Summary of the Invention

[0005] The primary objective of this invention is to provide a pharmaceutical preparation for treating atopic dermatitis.

[0006] A second objective of this invention is to provide a method for preparing a pharmaceutical preparation for treating atopic dermatitis.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A pharmaceutical preparation for treating atopic dermatitis, the pharmaceutical preparation comprising the following raw materials in parts by weight: 10-15 parts recombinant CFL1 protein, 1.5-3 parts seaweed polysaccharide, 18-22 parts sodium alginate, 10-16 parts poloxamer 407, 1-2 parts ascorbic acid, and 34-45 parts water; the amino acid sequence of the recombinant CFL1 protein is shown in SEQ ID NO.1.

[0008] Furthermore, the pharmaceutical preparation comprises the following raw materials in parts by weight: 12 parts recombinant CFL1 protein, 2.2 parts seaweed polysaccharide, 20 parts sodium alginate, 13 parts poloxamer 407, 1.5 parts ascorbic acid, and 40 parts water.

[0009] Furthermore, the preparation method of the *Hypericum perforatum* polysaccharide includes the following steps: (1) Take the spiny sea bream, remove the internal organs, wash and air dry, then crush and pass through a 20-mesh sieve to obtain sea bream powder; (2) Take the sea bream powder obtained in step (1), add acetone at a material-to-liquid ratio of 1:5 (w / v), heat and reflux at 55°C twice for 2–3 h each time, filter, discard the filtrate, dry the residue to obtain pretreated sea bream powder. (3) Take the pretreated dried powder of sea bream obtained in step (2), add it to 0.1 mol / L NaOH solution at a material-to-liquid ratio of 1:5 (w / v), stir and extract at 4℃ for 2–4 h, centrifuge, and take the supernatant; (4) Adjust the pH of the supernatant obtained in step (3) to 7.0 with hydrochloric acid, add 1% (w / v) Bacillus subtilis neutral protease, and enzymatically hydrolyze in a water bath at 45–50 ℃ for 3–4 h. Cool to room temperature, centrifuge at 4 ℃ to obtain the supernatant, and then obtain the extract of sea bream polysaccharide. (5) The extract of sea hyacinth polysaccharide obtained in step (4) was deproteinized and decolorized using Sevage reagent. It was then transferred to a dialysis bag with a molecular weight cutoff of 3500 Da for dialysis treatment. Subsequently, it was concentrated under reduced pressure and freeze-dried to obtain sea hyacinth polysaccharide.

[0010] Furthermore, the specific preparation steps of the recombinant CFL1 protein are as follows: 1) The amino acid sequence of recombinant CFL1 protein was designed by adding the artificial peptide GTFTSDYSSYLEGP to the end of the amino acid sequence from position 3 to 160 of wild-type CFL1, as shown in SEQ ID NO.1; 2) Based on the amino acid sequence of the recombinant CFL1 protein SEQ ID NO.1, a nucleotide sequence was designed in reverse and codons were optimized to obtain the nucleotide sequence encoding the recombinant CFL1 protein, as shown in SEQ ID NO.2; 3) Based on the obtained nucleotide sequence SEQ ID NO.2, design upstream and downstream primers to amplify the corresponding nucleotide sequence of the recombinant CFL1 protein, ligate the nucleotide sequence of the recombinant CFL1 protein amplified by the primers to the pET30a vector, and construct a recombinant plasmid. 4) Transform the recombinant plasmid constructed in step (3) into competent cells, induce expression with IPTG, and obtain cell pellet; 5) The bacterial precipitate obtained in step 4) was disrupted by sonication, and the supernatant was taken for separation and purification to obtain recombinant CFL1 protein.

[0011] Further, the upstream primer sequence of step 3) is shown in SEQ ID NO.3; the downstream primer sequence is shown in SEQ ID NO.4.

[0012] Further, in step 4), the competent cells are E. coli BL21 (DE3); the induction concentration of IPTG is 0.3-0.6 mM, and the induction time of IPTG is 6-10 h.

[0013] Furthermore, in step 5), the separation and purification is performed by affinity chromatography.

[0014] The preparation method of a pharmaceutical formulation for treating atopic dermatitis according to any one of the above is as follows: S1 Weigh out the recombinant CFL1 protein, sea bream polysaccharide, sodium alginate, poloxamer 407, ascorbic acid and water in the weight proportions mentioned above; S2 Add poloxamer 407 to deionized water accounting for 40%-50% of the total water volume, stir and swell at 2-8℃ for 12-24h to obtain poloxamer 407 gel matrix; S3 Add sodium alginate to deionized water accounting for 30%-40% of the total water volume, stir and swell at room temperature for 2-4 hours to obtain sodium alginate colloidal solution; S4. The poloxamer 407 gel matrix obtained in step S2 is mixed with the sodium alginate colloidal solution obtained in step S3, and stirred at 2-8℃ for 0.5-1 h to obtain the mixed matrix. S5 Add sea bream polysaccharide and ascorbic acid to the mixed matrix obtained in step S4, and stir at 2-8℃ until completely dissolved; S6 Dissolve the recombinant CFL1 protein in the remaining deionized water and slowly add it to the solution obtained in step S5. Stir gently at 50-100 rpm for 10-20 minutes at 2-8℃ to disperse it evenly. Let it stand at 2-8℃ to remove bubbles, and the drug preparation for treating atopic dermatitis is obtained.

[0015] Compared with the prior art, the main advantages of the present invention are as follows: This invention provides a pharmaceutical formulation for treating atopic dermatitis. A recombinant CFL1 protein is constructed and expressed using gene recombination technology. This recombinant protein possesses important functional sites of the original CFL1, thereby achieving efficient expression of CFL1 analogs and increasing its lipid solubility for easier skin absorption. It can regulate T-cell cytoskeleton remodeling, reduce the release of inflammatory factors, and repair skin barrier function. *Hypericum perforatum* polysaccharide can inhibit PAR-2 ​​receptor expression, reduce TSLP release, improve itching, and alleviate dermal capillary dilation, thereby relieving erythema and edema. The two components work synergistically to alleviate symptoms such as skin inflammation, edema, and scaling, promote skin tissue repair and normalization, reduce inflammatory responses, and effectively relieve itching behavior in mice with atopic dermatitis. This has significant implications for the treatment of atopic dermatitis and has promising market application prospects. Attached Figure Description

[0016] Figure 1 The plasmid map of recombinant plasmid pET30a-CFL1; Figure 2 This is the result of immunohistochemical staining; Figure 3 The level of the cytokine TNF-α in mouse serum; Figure 4 The level of the cytokine IL-4 in mouse serum; Figure 5 The effect of a drug formulation used to treat atopic dermatitis on the number of scratches per 30 minutes in each group of mice. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0018] The preparation method of the *Hylocereus undatus* polysaccharide used in this embodiment of the invention includes the following steps: (1) Take the spiny sea bream, remove the internal organs, wash and air dry, then crush and pass through a 20-mesh sieve to obtain sea bream powder; (2) Take the sea bream powder obtained in step (1), add acetone at a material-to-liquid ratio of 1:5 (w / v), heat and reflux at 55°C twice for 2 hours each time, filter, discard the filtrate, dry the residue to obtain pretreated sea bream powder. (3) Take the pretreated dried powder of sea bream obtained in step (2), add it to 0.1 mol / L NaOH solution at a material-to-liquid ratio of 1:5 (w / v), stir and extract at 4℃ for 3 h, centrifuge, and take the supernatant; (4) Adjust the pH of the supernatant obtained in step (3) to 7.0 with hydrochloric acid, add 1% (w / v) Bacillus subtilis neutral protease, and enzymatically hydrolyze in a water bath at 47 ℃ for 3 h. Cool to room temperature, centrifuge at 4 ℃ to obtain the supernatant, and then obtain the extract of sea bream polysaccharide.

[0019] (5) The polysaccharide extract of *Hydrocotyle vulgaris* obtained in step (4) was subjected to protein removal treatment twice using Sevage reagent. The aqueous phase was taken and hydrogen peroxide with a volume of 1 / 100 of the aqueous phase was added. The solution was decolorized to light yellow at 45°C. (6) The liquid obtained in step (5) is placed into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed for 32 hours, with the dialysis water being changed every 7 hours. (7) The dialysis solution was concentrated under reduced pressure and freeze-dried to obtain sea hyacinth polysaccharide.

[0020] Example 1 A pharmaceutical preparation for treating atopic dermatitis, the pharmaceutical preparation comprising the following raw materials in parts by weight: 12 parts recombinant CFL1 protein, 2.2 parts seaweed polysaccharide, 20 parts sodium alginate, 13 parts poloxamer 407, 1.5 parts ascorbic acid, and 40 parts water.

[0021] The specific preparation steps for the recombinant CFL1 protein are as follows: (1) The amino acid sequence of recombinant CFL1 protein was designed by adding the artificial peptide GTFTSDYSSYLEGP to the end of the amino acid sequence from position 3 to 160 of wild-type CFL1, as shown in SEQ ID NO.1.

[0022] (2) Based on the amino acid sequence of the recombinant CFL1 protein SEQ ID NO.1, the nucleotide sequence was reverse-engineered and the codons were optimized according to the codon preference of E. coli expressing heterologous proteins to obtain the nucleotide sequence encoding the recombinant CFL1 protein, as shown in SEQ ID NO.2.

[0023] (3) Based on the obtained nucleotide sequence SEQ ID NO.2, upstream and downstream primers for amplifying the corresponding nucleotide sequence of the recombinant CFL1 protein were designed. The upstream primer sequence is shown in SEQ ID NO.3, containing the Nde I restriction site CATATG; the downstream primer sequence is shown in SEQ ID NO.4, containing the Hind III restriction site AAGCTT. Using the nucleotide sequence SEQ ID NO.2 of the recombinant CFL1 protein as a template, the recombinant CFL1 protein gene was amplified by PCR, and the target fragment was purified using a gel extraction kit. The plasmid pET30a was double-digested with restriction endonucleases Nde I and Hind III, and the double digestion reaction system is shown in Table 2. The amplified and recovered CFL1 gene fragment was ligated with the target fragment of the expression vector pET30a using T4 DNA ligase to construct the recombinant plasmid pET30a-CFL1. The constructed recombinant plasmid pET30a-ELP3 was transformed into E. coli DH5α competent cells and cultured in LB agar plates containing 100 μg / mL kanamycin. Single colonies were picked for colony PCR identification, and the plasmid was extracted and sequenced to obtain the correct recombinant plasmid pET30a-CFL1. The plasmid map of recombinant plasmid pET30a-CFL1 is shown below. Figure 1 As shown.

[0024] Table 1 Sequence List Table 2. Double enzyme digestion reaction system (4) Transform the correct recombinant plasmid pET30a-CFL1 constructed in step (3) into E. coli BL21 (DE3) competent cells, spread it on LB plates containing 100 μg / mL kanamycin, and incubate overnight in an inverted 37°C incubator to obtain the recombinant strain, named pET30a-CFL1 / BL21.

[0025] (5) Select positive monoclonal colonies and inoculate them into LB broth containing 100 μg / mL kanamycin. Incubate overnight at 37°C with shaking to obtain seed culture. Inoculate the seed culture into LB broth containing 100 μg / mL kanamycin at a 5 v / v inoculation rate and incubate at 37°C with shaking to obtain OD. 600 The temperature was lowered to 25°C and 0.5 mM IPTG was added to induce recombinant CFL1 protein expression for 8 h.

[0026] (6) After induction, the bacterial cells were collected by centrifugation, and after sonication, they were centrifuged again to obtain the supernatant. The recombinant CFL1 protein was purified by affinity chromatography. The target protein eluent was collected and concentrated to a concentration of 1.0 mg / mL to obtain the recombinant CFL1 protein.

[0027] This embodiment also provides a method for preparing a pharmaceutical formulation for treating atopic dermatitis, the specific preparation method being as follows: S1 Weigh out the recombinant CFL1 protein, sea bream polysaccharide, sodium alginate, poloxamer 407, ascorbic acid and water in the weight proportions mentioned above; S2 Add poloxamer 407 to deionized water accounting for 45% of the total water volume, stir and swell at 5°C for 18 h to obtain poloxamer 407 gel matrix. S3 Add sodium alginate to deionized water accounting for 35% of the total water volume, stir and swell at room temperature for 3 hours to obtain sodium alginate colloidal solution; S4. The poloxamer 407 gel matrix obtained in step S2 is mixed with the sodium alginate colloidal solution obtained in step S3, and stirred at 4°C for 0.7 h to obtain the mixed matrix. S5 Add sea bream polysaccharide and ascorbic acid to the mixed matrix obtained in step S4, and stir at 4°C until completely dissolved; S6 Dissolve the recombinant CFL1 protein in the remaining deionized water and slowly add it to the solution obtained in step S5. Stir gently at 70 rpm for 15 min at 4°C to disperse it evenly. Let it stand at 2-8°C to remove bubbles, and the drug preparation for treating atopic dermatitis is obtained.

[0028] Example 2 A pharmaceutical preparation for treating atopic dermatitis, the pharmaceutical preparation comprising the following raw materials in parts by weight: 10 parts recombinant CFL1 protein, 1.5 parts seaweed polysaccharide, 18 parts sodium alginate, 10 parts poloxamer 407, 1 part ascorbic acid, and 34 parts water.

[0029] The preparation method of the recombinant CFL1 protein is the same as in Example 1.

[0030] This embodiment also provides a method for preparing a pharmaceutical formulation for treating atopic dermatitis, the specific preparation method being as follows: S1 Weigh out the recombinant CFL1 protein, sea bream polysaccharide, sodium alginate, poloxamer 407, ascorbic acid and water in the weight proportions mentioned above; S2 Add poloxamer 407 to deionized water accounting for 40% of the total water volume, stir and swell at 2℃ for 12 h to obtain poloxamer 407 gel matrix. S3 Add sodium alginate to deionized water accounting for 30% of the total water volume, stir and swell at room temperature for 2 hours to obtain sodium alginate colloidal solution; S4. The poloxamer 407 gel matrix obtained in step S2 is mixed with the sodium alginate colloidal solution obtained in step S3, and stirred at 2°C for 0.5 h to obtain a mixed matrix. S5 Add sea bream polysaccharide and ascorbic acid to the mixed matrix obtained in step S4, and stir at 2°C until completely dissolved; S6. After dissolving the recombinant CFL1 protein in the remaining deionized water, slowly add it to the solution obtained in step S5, and gently stir at 50 rpm for 10 min at 2°C to disperse it evenly; let it stand at 2°C to remove bubbles, and the drug preparation for treating atopic dermatitis is obtained.

[0031] Example 3 A pharmaceutical preparation for treating atopic dermatitis, the pharmaceutical preparation comprising the following raw materials in parts by weight: 15 parts recombinant CFL1 protein, 3 parts seaweed polysaccharide, 22 parts sodium alginate, 16 parts poloxamer 407, 2 parts ascorbic acid, and 45 parts water.

[0032] The preparation method of the recombinant CFL1 protein is the same as in Example 1.

[0033] This embodiment also provides a method for preparing a pharmaceutical formulation for treating atopic dermatitis, the specific preparation method being as follows: S1 Weigh out the recombinant CFL1 protein, sea bream polysaccharide, sodium alginate, poloxamer 407, ascorbic acid and water in the weight proportions mentioned above; S2 Add poloxamer 407 to deionized water accounting for 50% of the total water volume, stir and swell at 8°C for 24 h to obtain poloxamer 407 gel matrix. S3 Add sodium alginate to deionized water accounting for 40% of the total water volume, stir and swell at room temperature for 4 hours to obtain sodium alginate colloidal solution. S4. Mix the poloxamer 407 gel matrix obtained in step S2 with the sodium alginate colloidal solution obtained in step S3, and stir at 8°C for 1 h to obtain a mixed matrix. S5 Add sea bream polysaccharide and ascorbic acid to the mixed matrix obtained in step S4, and stir at 8°C until completely dissolved; S6. After dissolving the recombinant CFL1 protein in the remaining deionized water, slowly add it to the solution obtained in step S5, and gently stir at 100 rpm for 20 min at 8°C to disperse it evenly; let it stand at 8°C to remove bubbles, and the drug preparation for treating atopic dermatitis is obtained.

[0034] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the recombinant CFL1 (Cofilin-1) protein in a pharmaceutical formulation for treating atopic dermatitis was replaced with commercially available Cofilin-1 protein; otherwise, they were the same as in Example 1.

[0035] The amino acid sequence of commercially available Cofilin-1 protein is shown in SEQ ID NO.5.

[0036] SEQ ID NO.5: MASGVAVSDGVIKVFNDMKVRKSSTPEEVKKRKKAVLFCLSEDKKNIILEEGKEILVGDVGQTVDDPYATFVKMLPDKDCRYALYDATYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKKLTGIKHELQANCYEEVKDRCTLAEKLGGSAVISLEGKPL; Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the polysaccharide of *Hypericum perforatum*, a component of a pharmaceutical preparation for treating atopic dermatitis, is omitted; otherwise, they are the same as in Example 1.

[0037] Experimental Example 1 Skin lesions from clinically diagnosed atopic dermatitis patients (experimental group) and healthy controls (control group) were collected. After fixation in 4% paraformaldehyde and paraffin embedding, 4 μm thick serial sections were prepared. Sections were routinely dewaxed to water, and antigens were retrieved by high-pressure autoclaving with citrate buffer (pH 6.0). Endogenous peroxidase was blocked by incubation in 3% H2O2 for 10 min. Rabbit anti-human CFL1 polyclonal antibody (1:200) was added and incubated overnight at 4℃. The next day, secondary antibody was added and incubated at 37℃ for 30 min. DAB staining, hematoxylin counterstaining, dehydration, clearing, and mounting were performed. The sections were observed and photographed under an optical microscope. Results are as follows: Figure 2 As shown.

[0038] The results are as follows Figure 2 As shown, no obvious CFL1 positive staining was observed in the control group (healthy skin tissue) at magnifications of 5×, 10×, and 20× (Figures A, B, and C, marked with red boxes). In the experimental group (AD patient skin lesions), CFL1 positive staining areas were observed in multiple different fields of view (Figures a, d, and g) under 5× low magnification (marked in red). The positive signal was clearly visible under 10× magnification (Figures b, e, and h) (marked in red). Furthermore, under 20× high magnification (Figures c, f, and i), brownish-yellow granules were visible within the positive cells (indicated by red arrows). These results indicate that CFL1 is highly expressed in AD patient skin lesions, but extremely low in normal skin tissue.

[0039] Experimental Example 2 (1) Immortalized human keratinocytes (HaCaT) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were digested with trypsin and the cell density was adjusted to 1×10⁻⁶. 5Cells were seeded at a density of 2 mL / well in 6-well cell culture plates and cultured for 24 h to allow for cell adhesion and confluence to approximately 80%. The original culture medium was then discarded, and the cells were replaced with serum-free DMEM medium for 12 h for synchronization.

[0040] (2) The cells were divided into the following 5 groups: blank control group (routine culture without any stimulation), model group (recombinant human TNF-α with a final concentration of 10 ng / mL was added and induced for 24 h), low-dose group of sea hyacinth polysaccharide (10 ng / mL TNF-α and 0.1 μg / mL sea hyacinth polysaccharide were added at the same time), medium-dose group of sea hyacinth polysaccharide (10 ng / mL TNF-α and 1 μg / mL sea hyacinth polysaccharide were added at the same time), and high-dose group of sea hyacinth polysaccharide (10 ng / mL TNF-α and 10 μg / mL sea hyacinth polysaccharide were added at the same time).

[0041] (3) Cells in each group were cultured for another 24 h at 37℃ in a 5% CO2 incubator. Cells were collected, total RNA was extracted and reverse transcribed into cDNA. Using GAPDH as an internal reference gene, the relative expression levels of PAR-2 ​​mRNA and TSLP mRNA were detected by real-time quantitative PCR. The upstream primer sequence of PAR-2 ​​is shown in SEQ ID NO.6, and the downstream primer sequence is shown in SEQ ID NO.7; the upstream primer sequence of TSLP is shown in SEQ ID NO.8, and the downstream primer sequence is shown in SEQ ID NO.9. The primer sequence listing is shown in Table 3; the effect of *Hylocereus undatus* polysaccharide on TNF-α-induced PAR-2 ​​and TSLP mRNA expression in HaCaT cells is shown in Table 4.

[0042] Table 3 Sequence List Table 4. Effects of *Hylocereus undatus* polysaccharide on TNF-α-induced PAR-2 ​​and TSLP mRNA expression in HaCaT cells. The results are shown in Table 4, which presents the effects of *Hylocereus undatus* polysaccharide on TNF-α-induced PAR-2 ​​and TSLP mRNA expression in HaCaT cells. Table 4 shows that, compared with the blank control group, the relative expression levels of PAR-2 ​​mRNA and TSLP mRNA in HaCaT cells were significantly increased after TNF-α stimulation in the model group, indicating that TNF-α successfully induced the activation of the PAR-2 / TSLP pathway. Compared with the model group, the low, medium, and high doses of *Hylocereus undatus* polysaccharide all reduced the mRNA levels of PAR-2 ​​and TSLP to varying degrees, approaching the levels of the blank control group. These results indicate that *Hylocereus undatus* polysaccharide can simultaneously downregulate the gene expression of PAR-2 ​​and TSLP, key mediators of pruritus in cells, suggesting that it may play a role in alleviating atopic dermatitis-related pruritus by regulating the local inflammatory microenvironment of the skin.

[0043] Experimental Example 3 (1) Seventy SPF-grade male BALB / c mice, aged 5-6 weeks and weighing approximately 20±2g, were randomly divided into a blank group, a model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, and Comparative Example 2 group, with 10 mice in each group. The rearing environment was a 12-hour day-night cycle, with the room temperature controlled at 20-24℃ and the humidity controlled at 50-60%, and free access to food and water. The mice were acclimatized for 7 days before the experiment. On the 1st and 2nd days of the experiment, 50 μL of 1% DNCB solution (acetone: olive oil = 4:1, v / v) was evenly applied to the shaved area (2.5 cm × 2.5 cm) on the back of the neck of the mice for sensitization. The same method was used to reinforce sensitization once on the 2nd day. Starting from day 8, 30 μL of 0.5% DNCB solution was applied to the same area for stimulation, once every 3 days for a total of 4 times (days 8, 11, 14, and 17). The blank group was replaced with an equal volume of acetone-olive oil mixture instead of DNCB.

[0044] (2) Starting from day 5, the mice in the Examples 1-3 and Comparative Examples 1-2 were given a drug preparation for the treatment of atopic dermatitis on the skin lesions on the back of the neck. The blank group and the model group were not given any application. The dosage was 20 μL each time, once a day, for 14 consecutive days (until day 18). The general condition of the mice was observed daily during the administration period.

[0045] (3) 24 hours after the last stimulation (day 18), the skin lesions on the neck and back were scored blindly according to the scoring criteria in Table 5. Each of the four items (erythema, edema, epidermal exfoliation, and scaling) was scored (0-3 points), and the total score (0-12 points) was calculated. The data are expressed as mean ± standard deviation (the standard deviation is rounded to one decimal place), n = 10, and the results are shown in Table 6.

[0046] Table 5 Scoring Criteria Table 6. Effects of different drug formulations on skin lesion scores in each group of mice. The results are shown in Table 6, which illustrates the effects of different drug formulations on the skin lesion scores of mice in each group. As shown in Table 6, compared with Comparative Examples 1 and 2, the drug formulations prepared in Examples 1-3 of this invention for treating atopic dermatitis significantly reduced the skin lesion scores of mice in each group. This indicates that the drug formulations prepared in this invention for treating atopic dermatitis have significant therapeutic effects, collectively alleviating symptoms such as skin inflammation, edema, and scaling, thereby repairing the skin barrier and relieving inflammatory skin lesions.

[0047] (4) On the last day of the experiment, whole blood was collected from mice in each group and centrifuged at 5000 rpm for 10 min at 37°C to separate the supernatant serum. The levels of cytokines TNF-α and IL-4 in mouse serum were detected using an ELISA kit. The results are as follows: Figure 3 , Figure 4 As shown.

[0048] The results are as follows Figure 3 , Figure 4 The figure shows the serum levels of the cytokines TNF-α and IL-4 in mice. TNF-α is a pro-inflammatory cytokine that directly induces skin inflammation; IL-4 is a Th2 cytokine that promotes IgE-mediated allergic reactions. Figure 3 , Figure 4 It was found that, compared with the blank group, the levels of TNF-α and IL-4 in the model group mice were significantly increased after induction, exacerbating the inflammatory symptoms. After applying the drug preparation for treating atopic dermatitis prepared in this invention, compared with Comparative Examples 1 and 2, the drug preparations for treating atopic dermatitis prepared in Examples 1-3 of this invention could significantly inhibit the levels of TNF-α and IL-4 cytokines in mouse serum. This indicates that the drug preparation for treating atopic dermatitis prepared in this invention can downregulate the levels of TNF-α and IL-4 cytokines in serum, thereby alleviating atopic dermatitis in mice.

[0049] (5) After the last stimulation (day 18), mice in each group were placed individually in transparent observation boxes for 10 minutes to acclimatize. Then, the mice's scratching behavior on the lesion area of ​​the neck and back was recorded using a camera system within 30 minutes. Scratching or biting of the lesion area with the hind paws (continuous scratching until brief cessation was counted as one instance) was used as an indicator of itch. Two observers unaware of the group assignments counted the number of scratches within 30 minutes, and the average value was taken. The results are as follows: Figure 5 As shown.

[0050] The results are as follows Figure 5 The figure shows the effect of different drug formulations on the number of scratches in mice within 30 minutes in each group. Figure 5It can be seen that, compared with the control group, the number of scratches by mice in the model group was significantly increased, indicating that DNCB induction was successful. Compared with Comparative Examples 1 and 2, the number of scratches by mice in Examples 1-3 of this invention was significantly reduced. This indicates that the drug preparations prepared in Examples 1-3 of this invention can effectively alleviate itching behavior in mice with atopic dermatitis.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A pharmaceutical preparation for treating atopic dermatitis, characterized by, The pharmaceutical preparation comprises the following raw materials in parts by weight: 10-15 parts of recombinant CFL1 protein, 1.5-3 parts of seaweed polysaccharide, 18-22 parts of sodium alginate, 10-16 parts of poloxamer 407, 1-2 parts of ascorbic acid, and 34-45 parts of water; the amino acid sequence of the recombinant CFL1 protein is shown in SEQ ID NO.

1.

2. The pharmaceutical preparation for treating atopic dermatitis according to claim 1, wherein The pharmaceutical preparation comprises the following raw materials in parts by weight: 12 parts recombinant CFL1 protein, 2.2 parts seaweed polysaccharide, 20 parts sodium alginate, 13 parts poloxamer 407, 1.5 parts ascorbic acid, and 40 parts water.

3. The pharmaceutical preparation for treating atopic dermatitis according to claim 1, wherein The preparation method of the *Hylocereus undatus* polysaccharide includes the following steps: (1) Take the spiny sea bream, remove the internal organs, wash and air dry, then crush and pass through a 20-mesh sieve to obtain sea bream powder; (2) Take the sea bream powder obtained in step (1), add acetone at a material-to-liquid ratio of 1:5 (w / v), heat and reflux at 55°C twice for 2–3 h each time, filter, discard the filtrate, dry the residue to obtain pretreated sea bream powder. (3) Take the pretreated dried powder of sea bream obtained in step (2), add it to 0.1 mol / L NaOH solution at a material-to-liquid ratio of 1:5 (w / v), stir and extract at 4℃ for 2–4 h, centrifuge, and take the supernatant; (4) Adjust the pH of the supernatant obtained in step (3) to 7.0 with hydrochloric acid, add 1% (w / v) Bacillus subtilis neutral protease, and enzymatically hydrolyze in a water bath at 45–50 ℃ for 3–4 h. Cool to room temperature, centrifuge at 4 ℃ to obtain the supernatant, and then obtain the extract of sea bream polysaccharide. (5) The extract of sea hyacinth polysaccharide obtained in step (4) was deproteinized and decolorized using Sevage reagent. It was then transferred to a dialysis bag with a molecular weight cutoff of 3500 Da for dialysis treatment. Subsequently, it was concentrated under reduced pressure and freeze-dried to obtain sea hyacinth polysaccharide.

4. A pharmaceutical preparation for treating atopic dermatitis according to claim 1, characterized in that, The specific preparation steps for the recombinant CFL1 protein are as follows: 1) The artificial peptide GTFTSDYSSYLEGP was added to the end of the amino acid sequence from position 3 to 160 of wild-type CFL1 to design the amino acid sequence of recombinant CFL1 protein, as shown in SEQ ID NO.1; 2) Based on the amino acid sequence of the recombinant CFL1 protein SEQ ID NO.1, a nucleotide sequence was designed in reverse and codons were optimized to obtain the nucleotide sequence encoding the recombinant CFL1 protein, as shown in SEQ ID NO.2; 3) Based on the obtained nucleotide sequence SEQ ID NO.2, design upstream and downstream primers to amplify the corresponding nucleotide sequence of the recombinant CFL1 protein, ligate the nucleotide sequence of the recombinant CFL1 protein amplified by the primers to the pET30a vector, and construct a recombinant plasmid. 4) Transform the recombinant plasmid constructed in step (3) into competent cells, induce expression with IPTG, and obtain cell pellet; 5) The bacterial precipitate obtained in step 4) was disrupted by sonication, and the supernatant was taken for separation and purification to obtain recombinant CFL1 protein.

5. A pharmaceutical preparation for treating atopic dermatitis according to claim 4, characterized in that, The upstream primer sequence of step 3) is shown in SEQ ID NO.3; the downstream primer sequence is shown in SEQ ID NO.

4.

6. A pharmaceutical preparation for treating atopic dermatitis according to claim 4, characterized in that, In step 4), the competent cells are E. coli BL21 (DE3); the induction concentration of IPTG is 0.3-0.6 mM, and the induction time of IPTG is 6-10 h.

7. A pharmaceutical preparation for treating atopic dermatitis according to claim 4, characterized in that, In step 5), the separation and purification process is performed using affinity chromatography.

8. A method for preparing a pharmaceutical preparation for treating atopic dermatitis according to any one of claims 1-7, characterized in that, The specific preparation method is as follows: S1 Weigh the recombinant CFL1 protein, sea hyacinth polysaccharide, sodium alginate, poloxamer 407, ascorbic acid and water according to the weight proportions described in claim 1; S2 Add poloxamer 407 to deionized water accounting for 40%-50% of the total water volume, stir and swell at 2-8℃ for 12-24 h to obtain poloxamer 407 gel matrix. S3 Add sodium alginate to deionized water accounting for 30%-40% of the total water volume, stir and swell at room temperature for 2-4 hours to obtain sodium alginate colloidal solution; S4. The poloxamer 407 gel matrix obtained in step S2 is mixed with the sodium alginate colloidal solution obtained in step S3, and stirred at 2-8℃ for 0.5-1 h to obtain the mixed matrix. S5 Add sea bream polysaccharide and ascorbic acid to the mixed matrix obtained in step S4, and stir at 2-8℃ until completely dissolved; S6 Dissolve the recombinant CFL1 protein in the remaining deionized water and slowly add it to the solution obtained in step S5. Stir gently at 50-100 rpm for 10-20 minutes at 2-8℃ to disperse it evenly. Let it stand at 2-8℃ to remove bubbles, and the drug preparation for treating atopic dermatitis is obtained.

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