Application of SjP40 protein, composition for promoting skin tissue repair, preparation method of composition and pharmaceutical preparation

Abstract

The invention relates to the technical field of biological medicine, and particularly provides application of SjP40 protein, a composition for promoting skin tissue repair, a preparation method of the composition and a pharmaceutical preparation. The SjP40 protein from schistosoma japonicum katsurada is applied to the field of skin injury repair, the SjP40 can remarkably accelerate wound closure, reduce the inflammatory cell infiltration level and enhance capillary and appendage regeneration, and the overall repair quality is superior to that of an existing treatment method. Meanwhile, the SjP40 protein is combined with the hydrogel, so that local controlled release and stable release of the hydrogel on a wound surface are realized, the problem of rapid degradation of protein activity in vitro is solved, and the bioavailability and the treatment efficiency of the hydrogel are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of SjP40 protein. It also relates to a composition for promoting skin tissue repair, its preparation method, and a pharmaceutical formulation. Background Technology

[0002] As the largest physical barrier organ in the human body, the skin plays a crucial role in resisting external damage and pathogen invasion. Common skin defects caused by trauma, surgical incisions, chronic diseases (such as diabetes and pressure ulcers), or infections often involve persistent inflammatory responses, impaired angiogenesis, and abnormal extracellular matrix remodeling during the repair process. For wounds involving full-thickness injuries or metabolic diseases, existing treatment methods have clear limitations: routine dressing changes are insufficient to effectively regulate the wound microenvironment; growth factor preparations (such as EGF and bFGF) are easily diluted by exudate, leading to reduced activity, and are also costly; physical therapies such as negative pressure wound therapy are complex to perform and insufficiently applicable to superficial wounds.

[0003] Studies have found that SjP40 protein, an immunomodulatory molecule derived from Schistosoma japonicum, can inhibit excessive inflammatory responses by inducing macrophage polarization towards the M2 anti-inflammatory phenotype. It can also regulate the TGF-β / Smad signaling pathway to alleviate tissue fibrosis and promote vascular endothelial cell migration and angiogenesis. However, current research mainly focuses on the application of SjP40 in liver fibrosis and immune diseases, with no reports on its application in skin repair. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides the application of SjP40 protein in the preparation of drugs or skin care products for skin tissue repair. It also provides an SjP40 protein hydrogel composition that can promote skin tissue repair, accelerate wound closure, reduce inflammatory cell infiltration levels, and enhance capillary and appendage regeneration.

[0005] In a first aspect, the present invention provides the application of SjP40 protein in the preparation of drugs or skin care products for skin tissue repair.

[0006] Preferably, the skin tissue repair includes repair of inflammatory wounds, knife wounds, bedsores, ulcers caused by diabetic complications, burns, scalds, frostbite, radiation-induced skin damage, healing of postoperative infected incisions, or promotion of growth after skin grafting.

[0007] Preferably, the dosage form of the drug or skin care product includes gel, liquid, cream, ointment, lotion, spray, injection, injection or poultice.

[0008] In a second aspect, the present invention provides a composition for promoting skin tissue repair, comprising SjP40 protein and Pluronic F127 hydrogel.

[0009] Preferably, the hydrogel, in addition to Pluronic F127 hydrogel, may also be selected from at least one of gelatin-sodium alginate composite hydrogel, chitosan hydrogel, sodium hyaluronate hydrogel, carbomer gel, and chitosan / gelatin / β-glycerophosphate composite hydrogel.

[0010] Preferably, the composition further includes at least one of growth factors, antimicrobial peptides, silver ions, antioxidants, stem cell-derived exosomes, or hemostatic agents.

[0011] In this invention, SjP40 protein can also be used in conjunction with the following bioactive substances to construct a multifunctional synergistic repair system: (1) when used in combination with growth factors (such as bFGF and EGF), it can improve the rate of epithelial reconstruction; (2) when used in combination with antimicrobial peptides or silver ions, it can enhance the anti-infection ability; (3) when combined with antioxidants (such as vitamin C and glutathione), it is suitable for high stress or chronic wounds; (4) when combined with stem cell-derived exosomes, it can be used for the repair of severe tissue defects; (5) when used in combination with hemostatic agents, it can be used for postoperative or bleeding wounds.

[0012] Preferably, the mass fraction of the Pluronic F127 hydrogel is 20% to 30%. More preferably, the mass fraction of the Pluronic F127 is 26%.

[0013] Preferably, the concentration of SjP40 protein in the composition is 0.3~0.7 mg / mL. More preferably, the concentration of SjP40 protein is 0.5 mg / mL.

[0014] This invention combines SjP40 protein with Pluronic F127 (PF127) hydrogel to achieve localized controlled and stable release of SjP40 protein at the wound site, overcoming the problem of rapid degradation of protein activity in vitro and improving its bioavailability and therapeutic efficiency. The PF127 hydrogel can rapidly gel at body temperature, forming a soft protective layer that effectively isolates external contamination while providing a moist environment, further promoting wound healing. Animal model experiments have confirmed that the SjP40+PF127 composition constructed in this invention significantly outperforms SjP40 protein alone or hydrogel alone in multiple indicators such as wound healing speed, tissue reconstruction integrity, and the degree of inflammatory cell infiltration, demonstrating a significant synergistic effect. This composite design not only possesses dual functions of anti-inflammation and repair but also significantly improves the stability and actual efficacy of protein drugs in in vitro applications.

[0015] Preferably, in addition to PF127 hydrogel, SjP40 protein can also be compounded with the following medical hydrogel systems to form topical gel compositions with different physicochemical properties and sustained-release characteristics. Specifically: compounded with gelatin-sodium alginate composite hydrogel, it can promote cell adhesion and tissue growth; compounded with chitosan hydrogel, it has natural antibacterial and hemostatic effects; compounded with sodium hyaluronate hydrogel, it has moisturizing and biocompatibility; compounded with carbomer gel, it is a classic drug carrier and its rheological properties are easily controlled; compounded with chitosan / gelatin / β-glycerophosphate composite hydrogel, it can control the gel temperature and degradation rate. In practical applications, the hydrogel system can be flexibly selected according to the different skin conditions, wound depths, and healing speed requirements of different patients.

[0016] Thirdly, the present invention provides a method for preparing the above composition, comprising the following steps: mixing 20% ​​to 30% by mass of Pluronic F127 hydrogel with SjP40 protein to obtain the composition; wherein the concentration of SjP40 protein in the composition is 0.3 to 0.7 mg / mL.

[0017] Fourthly, the present invention provides a pharmaceutical preparation comprising the above-described composition, wherein the pharmaceutical preparation is a topical gel, patch, or spray.

[0018] Preferably, the composition can be made into the following different delivery systems according to different clinical application scenarios: (1) Microneedle hydrogel system: SjP40 is directly delivered to the dermis through polymer microneedle structure, which enhances penetration and improves protein bioavailability; (2) Patch-type hydrogel dressing: The gel is loaded onto the support membrane material to make a flexible dressing suitable for large-area wound management; (3) Spray-type thermosensitive gel: The wound is quickly covered by low-temperature spraying, and gelation is triggered by body temperature to form a sterile protective film; (4) Injectable in vivo gel system: It is applied to deep skin tissue or subcutaneous postoperative wound cavity, and gelation is triggered by body temperature to reduce exudation and scarring.

[0019] Preferably, the composition can be designed into the following dosage forms to suit different clinical application scenarios: (1) fluid gel for irregular wounds; (2) gel patch for large-area chronic wounds; (3) hydrogel beads and microsphere system for easy precision management; (4) composite nanogel for transdermal systems; (5) freeze-dried dressing / film for easy transportation and storage.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to apply SjP40 protein derived from Schistosoma japonicum to the field of skin injury repair, breaking through its original limitations in anti-inflammatory and anti-fibrotic applications and broadening its biomedical application boundaries. Experimental results show that SjP40 can significantly accelerate wound closure, reduce the level of inflammatory cell infiltration, and enhance capillary and appendage regeneration. It has unique advantages in promoting wound healing, inhibiting inflammation, promoting angiogenesis and tissue remodeling, and achieving repair quality superior to existing treatment methods.

[0021] (2) This invention constructs a topical gel formulation with good controlled-release function and tissue adhesion properties. PF127 is liquid at low temperatures, which facilitates protein encapsulation; upon contact with body temperature, it rapidly gels, forming a protective layer at the wound site and continuously releasing SjP40. This design not only improves protein stability and local utilization, but also enhances the residence time of the formulation at the wound site and the duration of treatment, belonging to a novel combination of protein-gel synergistic therapy.

[0022] (3) The hydrogel composition containing SjP40 protein provided by the present invention can be easily prepared under low temperature conditions without complex processes. It can be made into topical gels, patches or spray products, etc., and has strong scalability. It is suitable for different types of skin wounds (such as postoperative wounds, abrasions, diabetic foot, etc.) and has high clinical translational value and market application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The image shows the wound outline of mice in each group in the experimental examples of the effectiveness of this invention; Figure 2 Line graphs showing the change in wound healing rate of mice in each group as a function of injury time in the experimental examples of the efficacy of this invention; Figure 3 The images show the HE staining results of mouse wounds in each group in the experimental examples of the efficacy of this invention; where (a) is the PBS group, (b) is the SjP40 group, (c) is the PF127 group, and (d) is the SjP40+PF127 group. Figure 4 The images show the Masson staining results of mouse wounds in each group in the experimental examples of the efficacy of this invention; where (a) is the PBS group, (b) is the SjP40 group, (c) is the PF127 group, and (d) is the SjP40+PF127 group. Figure 5The graph shows the collagen volume fraction of each mouse wound in the experimental cases of the present invention. Detailed Implementation

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the specific embodiments will be briefly introduced below. Obviously, the embodiments described below are some embodiments of the present invention. For those skilled in the art, other embodiments can be obtained based on these embodiments without creative effort.

[0026] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials or instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0027] Example 1 This embodiment provides an SjP40 protein hydrogel composition, which is prepared from SjP40 protein and Pluronic F127 hydrogel (abbreviated as PF127 hydrogel). The mass fraction of PF127 hydrogel is 26%, and the concentration of SjP40 protein in the composition is 0.5 mg / mL.

[0028] The SjP40 protein used in this embodiment was synthesized by Beijing BGI Protein Research Center Co., Ltd.

[0029] The preparation process of the SjP40 protein hydrogel composition provided in this embodiment is as follows: Pluronic F127 was added to deionized water and magnetically stirred until completely dissolved to prepare a PF127 hydrogel with a mass fraction of 26%. SjP40 protein was added to the PF127 hydrogel and then mixed at 4°C to form an SjP40 protein hydrogel composition with a concentration of 0.5 mg / mL.

[0030] Example 2 This embodiment provides an SjP40 protein hydrogel composition, which is prepared from SjP40 protein and Pluronic F127 hydrogel (abbreviated as PF127 hydrogel). The mass fraction of PF127 hydrogel is 20%, and the concentration of SjP40 protein in the composition is 0.3 mg / mL.

[0031] The SjP40 protein used in this embodiment was synthesized by Beijing BGI Protein Research Center Co., Ltd.

[0032] The preparation process of the SjP40 protein hydrogel composition provided in this embodiment is as follows: Pluronic F127 was added to deionized water and magnetically stirred until completely dissolved to prepare a PF127 hydrogel with a mass fraction of 20%. SjP40 protein was added to the PF127 hydrogel and then mixed at 4°C to form an SjP40 protein hydrogel composition with a concentration of 0.3 mg / mL.

[0033] Example 3 This embodiment provides an SjP40 protein hydrogel composition, which is prepared from SjP40 protein and Pluronic F127 hydrogel (abbreviated as PF127 hydrogel). The mass fraction of PF127 hydrogel is 30%, and the concentration of SjP40 protein in the composition is 0.7 mg / mL.

[0034] The SjP40 protein used in this embodiment was synthesized by Beijing BGI Protein Research Center Co., Ltd.

[0035] The preparation process of the SjP40 protein hydrogel composition provided in this embodiment is as follows: Pluronic F127 was added to deionized water and magnetically stirred until completely dissolved to prepare a PF127 hydrogel with a mass fraction of 30%. SjP40 protein was added to the PF127 hydrogel and then mixed at 4°C to form an SjP40 protein hydrogel composition with a concentration of 0.7 mg / mL.

[0036] Effect Experiment Example The effects of different dressings on the healing of skin lesions in mice were investigated, and the specific procedures are as follows: 1. Establishment of a skin injury model: C57 strain mice were selected, which are similar in species, age, and physique. The backs of the mice were shaved and disinfected with iodine. A standardized circular wound was created on the back using a 5mm diameter sterile punch to avoid damaging the muscle layer. After disinfection, the wound was immediately covered with a Tegaderm bandage for fixation to increase skin tension and prevent skin wrinkling around the mouse wound from affecting healing, thus simulating the human skin wound healing process.

[0037] 2. Grouping and drug administration: Twenty-four mice were randomly divided into four groups, and the drug was applied topically once daily for seven consecutive days. Control group (PBS group): 20 μL of sterile saline was applied topically to each wound. SjP40 group: Apply 20μL of SjP40 protein aqueous solution (0.5mg / mL) to each wound. PF127 group: 20 μL of PF127 hydrogel with a mass fraction of 26% was applied topically to each wound. SjP40+PF127 group: Apply 20 μL of the SjP40 protein hydrogel composition provided in Example 1 of this invention to each wound.

[0038] 3. Wound observation and assessment: (1) Monitoring of wound healing rate: Wound images were taken at a fixed distance on postoperative days 0, 2, 4, 6, 8, and 10, and the healing rate was calculated. Healing rate = (Initial area - Unhealed area) / Initial area × 100%; The wound outlines of each group on days 0, 2, 4, 6, 8, and 10 are shown in the following figures. Figure 1 As shown in the figure, the line graphs depicting the changes in wound healing rate over time for each group are as follows: Figure 2 As shown. By Figure 1 and Figure 2 It was found that all wounds healed completely within approximately 14 days post-surgery. However, on days 2, 4, 6, and 10, the healing rates of the SjP40 group, PF127 group, and SjP40+PF127 group were significantly higher than those of the PBS group. Among these, the PF127+SjP40 group showed the most significant increase in wound healing rate, while there was no significant difference in wound healing rate between the SjP40 group and the PF127 group (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). Statistical analysis was performed using one-way ANOVA. Brown-Forsythe multiple comparisons were used to compare the treatment groups at different time points. Error bars represent the mean ± standard error.

[0039] (2) Histopathological examination: On the 10th day after surgery, wound tissue from each group was taken for HE staining to assess the pathological state of the skin wounds in each group. The HE staining results for each group are as follows: Figure 3 As shown, where, Figure 3 (a) is the PBS group. Figure 3 (b) is group SjP40. Figure 3 (c) is group PF127. Figure 3 (d) represents the SjP40+PF127 group. In the figure, the dashed line indicates the skin wound area, "▲" indicates the epidermis, "↓" indicates the hair follicle, and "→" indicates the sebaceous gland. Figure 3As can be seen, the dashed line represents the skin wound area. Compared with the PBS group, the inflammatory cell infiltration in the dermis of the wound area of ​​the SjP40 group, PF127 group, and PF127+SjP40 group was significantly reduced. "▲" represents the epidermis. The epidermis of the wound area of ​​the mice in the PBS group was significantly thicker than that of the surrounding normal skin area, while this was not obvious in the SjP40 group, PF127 group, and PF127+SjP40 group. "↓" represents hair follicles, and "→" represents sebaceous glands. Compared with the PBS group, a small number of skin appendages such as hair follicles and sebaceous glands can be seen in the wound area of ​​the SjP40 group and PF127 group. However, a large number of skin appendages have formed in the wound area of ​​the PF127+SjP40 group by day 10, and the dermal structure of the skin wound is similar to that of the normal skin around the wound.

[0040] On postoperative day 10, wound tissue samples from each group were collected for Masson staining to assess collagen fiber deposition in the wound area. The collagen area in each wound area was quantified using ImageJ software. Multiple images were taken along the wound bed for each sample in each group, and the average values ​​were compared. The Masson staining results for each group are shown below. Figure 4 As shown, red represents muscle fibers, and blue represents collagen fibers. Among them, Figure 4 (a) is the PBS group. The blue collagen fibers in the wound area are lighter in color and sparsely arranged, indicating less collagen deposition. Figure 4 (b) For the SjP40 group, compared with the PBS group, the blue collagen fibers were darker and the density increased, indicating that collagen deposition was promoted. Figure 4 (c) is the PF127 group. Compared with the PBS group, the coloring and density of blue collagen fibers have increased to a certain extent. Figure 4 (d) is the SjP40+PF127 group. The blue collagen fibers in the wound area are the darkest and have the highest density. The fibers are also more regularly arranged, indicating that the collagen deposition is the most significant.

[0041] The collagen volume fraction graphs for each group are shown below. Figure 5 As shown, compared with the PBS group, the collagen volume fraction in the wound area of ​​the SjP40 group, PF127 group, and PF127+SjP40 group was significantly increased, with the PF127+SjP40 group showing the most significant increase. However, there was no significant difference in collagen volume fraction between the wound areas of the SjP40 group and the PF127 group (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). Statistical analysis was performed using one-way ANOVA, and comparisons were made using Brown-Forsythe multiple comparisons.

[0042] 4. Results Analysis: The wound healing rate of the SjP40+PF127 group was significantly higher than that of other groups. HE section analysis showed that the SjP40+PF127 group had the least inflammatory infiltration, no significant thickening of the epidermis, and good recovery of appendages such as hair follicles and sebaceous glands in the dermis. Therefore, the combination of SjP40 protein and PF127 hydrogel can significantly accelerate wound closure, reduce inflammatory cell infiltration, promote collagen fiber deposition and skin appendage regeneration, and help reduce post-healing scar hyperplasia. Its repair effect is superior to that of using SjP40 protein or PF127 hydrogel alone, demonstrating a synergistic effect.

[0043] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Application of SjP40 protein in the preparation of drugs or skin care products for skin tissue repair.

2. The application as described in claim 1, characterized in that, The skin tissue repair includes the repair of inflammatory wounds, knife wounds, bedsores, ulcers caused by diabetic complications, burns, scalds, frostbite, radiation-induced skin damage, healing of postoperative infected incisions, or promoting growth after skin grafting.

3. The application as described in claim 1, characterized in that, The dosage forms of the drugs or skin care products include gels, liquids, creams, ointments, lotions, sprays, injections, needles, or poultices.

4. A composition for promoting skin tissue repair, characterized in that, Including SjP40 protein and Pluronic F127 hydrogel.

5. The composition according to claim 4, characterized in that, The composition further includes at least one of growth factors, antimicrobial peptides, silver ions, antioxidants, stem cell-derived exosomes, or hemostatic agents.

6. The composition according to claim 4, characterized in that, The mass fraction of the Pluronic F127 hydrogel is 20%~30%.

7. The composition according to claim 4, characterized in that, In the composition, the concentration of SjP40 protein is 0.3~0.7 mg / mL.

8. A method for preparing the composition according to any one of claims 4-7, characterized in that, Includes the following steps: The composition was obtained by mixing 20% ​​to 30% by mass of Pluronic F127 hydrogel with SjP40 protein; the concentration of SjP40 protein in the composition was 0.3 to 0.7 mg / mL.

9. A pharmaceutical formulation comprising the composition according to any one of claims 4-7, characterized in that, The pharmaceutical preparation is in the form of a topical gel, patch, or spray.

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