CN115991758B - Pearl powder polypeptide for promoting diabetic wound healing and application thereof - Google Patents

Abstract

The invention discloses a small molecular polypeptide derived from pearl powder and application thereof in preparing a medicament or an external dressing for promoting diabetic wound healing, and belongs to the technical field of medicines. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1; or the polypeptide is a conservative variant obtained by deleting, replacing, inserting or/and adding one to several amino acids on the basis of the amino acid sequence shown in SEQ ID NO. 1. The polypeptide provided by the invention has biological activity for promoting healing of acute and chronic skin wounds and mucosal ulcers, has good application prospect, and can be used for preparing external medicines or external dressings for promoting healing of diabetic wounds.

Description

A kind of pearl powder polypeptide that promotes diabetic wound healing and its application

Technical field

The invention belongs to the field of medical technology. Specifically, it relates to a small molecule polypeptide derived from pearl powder and its application in the preparation of drugs or external dressings for promoting diabetic wound healing.

Background technique

Among the many complications of diabetes, impaired wound healing in diabetic patients is a typical complication of diabetes and the main cause of non-traumatic lower limb amputation. This complication increases the lifetime risk of chronic foot ulcers in diabetics to as high as 32%. In the past few decades, with the continuous development of various new drugs, new technologies and new treatment strategies, especially the rise of multidisciplinary comprehensive treatment models in recent years, the therapeutic effect of diabetic wounds has been improved to some extent, but the treatment cycle is long and Problems such as low short-term wound healing rates still cannot be fundamentally solved. Therefore, actively exploring new therapeutic drugs and methods to shorten wound healing time is of great significance for improving patients' quality of life and reducing medical costs.

As a traditional Chinese medicine, pearl powder has been used among Chinese people for thousands of years. In traditional applications, the topical use of pearl powder can treat a variety of acute and chronic wounds that do not heal. It is currently included in the "Pharmacopoeia of the People's Republic of China". Modern clinical observations show that pearl powder alone or in combination can significantly promote the healing of various types of acute and chronic skin wounds and mucosal ulcers, including peptic ulcers, oral ulcers, skin injuries, wound ulcers, bedsores, and burns. In vivo and in vitro animal cell experiment results show that the water-soluble components of pearl powder have a significant healing effect on animal wound injury models. They can exert antioxidant effects by regulating ROS levels and promote the migration of skin fibroblasts, thereby accelerating wound healing. In addition, pearl powder is involved in regulating cell growth and repair-related signals that are closely related to wound healing, including stimulating fibroblast mitosis, enhancing cell adhesion, promoting collagen deposition, and inhibiting metalloproteinase (TIMP)-1 activity.

Existing research shows that in addition to containing a large amount of inorganic components such as calcium carbonate and trace elements, pearl powder also contains rich organic components such as active peptides. CN113559240A discloses a pearl polypeptide liquid and its application in promoting wound healing. The separation and extraction process of the pearl polypeptide liquid includes the following steps: mixing pearl ultrafine powder with 1±0.5wt% PBS solution, pearl ultrafine powder and PBS solution The dosage ratio is 1: (4±0.5)g/mL. Use a supercritical carbon dioxide extraction instrument to extract small molecule peptides from pearl powder. Collect the extracted mixture and centrifuge it in a high-speed centrifuge at 5000±1000rpm for 10-15min to collect the upper liquid. , which is pearl peptide liquid. However, although this patented technology obtains a pearl powder polypeptide liquid through extraction and separation technology, the polypeptide liquid is a mixture of multiple small molecule peptides, including inactive or low-activity small molecule peptides. Based on this, we conducted a screening study on pearl powder peptides that promote the activity of chronic diabetic wounds. The results found that the small molecule peptide Na-1 derived from pearl powder has a good effect on promoting the healing of diabetic wounds.

Contents of the invention

In view of the shortcomings of the existing technology, the first object of the present invention is to provide a highly active polypeptide derived from pearl powder and capable of promoting diabetic wound healing.

The above objects of the present invention are achieved through the following technical solutions: a polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1; or the polypeptide is based on the amino acid sequence shown in SEQ ID NO.1 Conservative variants obtained by deletion, substitution, insertion or/and addition of conservative mutations of one to several amino acids.

The amino acid sequence of SEQ ID NO. 1 is ISKCFIACGIGQRQSPINIV, and the polypeptide is named Na-1. The polypeptide Na-1 provided by the present invention can be customized directly from a commercial peptide synthesis company, or can be synthesized using a commercially available automatic synthesizer according to the manufacturer's operating procedures.

On the other hand, the present invention also provides the use of the polypeptide in preparing drugs or external dressings for promoting diabetic wound healing.

On the other hand, the present invention also provides a composition for promoting diabetic wound healing, which composition contains the above-mentioned polypeptide.

Further preferably, the composition for promoting diabetic wound healing as described above is a gel. Still further preferably, the gel agent includes the polypeptide, a gel matrix, a moisturizing agent, a penetration enhancer, a pH adjuster and water.

It should be noted that the gelling matrix is selected from one or a mixture of two or more carbomers, hydroxypropyl methylcellulose, and sodium hyaluronate; the moisturizing agent is glycerin; the accelerator The penetrating agent is azone; the pH regulator is sodium hydroxide. In addition, the gel may also contain a preservative, and the preservative may be one or more selected from the group consisting of potassium sorbate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate and sodium benzoate.

Compared with the existing technology, the polypeptides provided by the present invention have biological activity that promotes the healing of acute and chronic skin wounds and mucosal ulcers, and have good application prospects. They can be used to prepare external drugs or external dressings that promote diabetic wound healing.

Description of the drawings

Figure 1 shows the healing process of total skin resection wound in diabetic rats. The wound healing status was recorded on days 0, 2, 4, 6, 8, 10, 12 and 14 days.

Figure 2 is a time course graph of the healing rate of total skin resection wounds in diabetic rats.

Figure 3 shows the H&E and Masson staining pictures of wounds in each group 14 days after modeling.

Figure 4 is a picture of Na-1 external gel.

Detailed ways

In order to better illustrate the characteristics and essence of the present invention, the pharmacological test results of Na-1 in promoting the healing of total thickness resection wounds in diabetic rats are provided below in the form of examples to illustrate its use in the field of medical technology. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. In addition, if the specific technical operation steps or conditions are not specified in the examples, the techniques or conditions described in the literature in the field or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

Example 1: Source and artificial synthesis of Na-1

Na-1 amino acid sequence: ISKCFIACGIGQRQSPINIV

Na-1 is a polypeptide sequence shared by multiple pearl proteins. The detailed information is shown in Table 1. Shanghai Sangon was commissioned to synthesize Na-1 for observation of the healing effect of diabetic wounds.

Table 1: Position information of Na-1 in the pearlin sequence

Example 2: Preparation of Na-1 external gel

Weigh carbomer (0.4051g) and hydroxypropyl methylcellulose (0.1031g) and slowly add them to 60 mL of distilled water, stirring while adding, until completely dissolved. Weigh an appropriate amount of sodium hydroxide and add it to the above solution, stir while adding, adjust the pH to 10, and obtain a colorless and transparent carbomer hydroxypropyl methylcellulose gel. Then add glycerin (5.2g) and stir evenly to obtain a gel matrix. Weigh Na-1 (0.12496g), add 20 mL of water to disperse, and obtain a Na-1 solution. Add the Na-1 solution to the gel matrix and stir evenly. Finally, add azone (1.1g) and make up the weight to 100g with distilled water. Stir evenly to obtain Na-1 external gel. This product is a milky white gel with stable properties, no stratification, no oil leakage, fine texture, colorless and transparent, suitable viscosity, easy to apply and clean, and no greasy feeling (Figure 4).

Example 3: Experiment on the effect of Na-1 on wound healing after total skin resection in diabetic rats

1. Preparation of Diabetic Rats

Male SD rats were intraperitoneally injected with 1% streptozotocin (streptozotocin dissolved in citric acid buffer with pH 4.5) 65mg/kg, 6mL/kg, and 72 hours after the injection of streptozotocin. For the first time, blood was taken from the tip of the tail to detect the random blood sugar value of the rats. The blood sugar value exceeded 16.7mmol/L as the standard for diabetic rats. Rats with blood sugar values greater than 16.7mmol/L were selected as experimental subjects. After stabilization for 5 days, Make wounds.

2. Preparation of wounds in diabetic rats

The rat was anesthetized with ether and fixed on the operating table in a prone position. Use an electric pusher to remove the long hair on the back of the rat, and then apply depilatory cream. After 6 minutes, scrape off the depilatory cream, wipe the remaining depilatory cream in the depilated area with warm water, and then use gauze. Dry, disinfect with iodine, lay out a surgical towel, cover the surgical area with a 10-cent coin (diameter 1.8cm) that has been sterilized by autoclaving, apply pressure to form a circular trace, use surgical scissors to press the trace and cut out the circular skin to Deep fascial layer. Since the thickness and tension of the skin on the back of each rat are different, in order to avoid misjudgment of the experimental results due to differences between the wounds, the number and location of the wounds prepared for each rat were designed: 2 wounds were prepared for each rat, It is located 1.5cm on the left and right sides of the spine. The distance between the center of the wound line on the same side and the neck is half the total length of the back.

3. Wound grouping and drug administration

There were 2 normal rats with a total of 4 wounds, which were set as the non-diabetic wound blank group (Normal group), which was the natural wound healing group of rats without any treatment. There were 8 diabetic rats with a total of 16 wounds. The wounds were numbered and grouped according to the random number table, and were divided into diabetic wound blank group (Model group), diabetic wound treated with epidermal growth factor group (EGF group, positive control), and diabetic wound. A total of 3 groups were given to the Na-1 group (Na-1 group); among them: Model group, 4 wounds, which were the natural wound healing group of diabetic rats, without any treatment; EGF group, 4 wounds, were the positive control drug EGF In the intervention group, medication was started on the day the wound was created. A certain amount of EGF gel (trade name: Yifu, specification: 50,000 IU (100 μg)/10g/tube, produced by Guilin Huanovi Gene Pharmaceutical Co., Ltd., approved by the National Drug Administration S20020112) was evenly applied to the wounds of diabetic rats until the wounds were completely covered; the Na-1 group, with 4 wounds, was the small molecule peptide Na-1 intervention group. Administration began on the day the wounds were created, and a certain amount of Na- 1. Gel (prepared in Example 2) was evenly applied to the wound surface of diabetic rats until the wound surface was just completely covered. Administer once a day at 10:00-12:00 in the morning, with free access to water and food. Take photos on days 0, 2, 4, 6, 8, 10, 12 and 14 after surgery and calculate the wound area at each time point. Calculate the healing rate according to the following formula: Healing rate (%) = (original area of the wound - area at each time point) )÷original wound area×100%. After 14 days, wound tissues from each group were taken, fixed with 4% paraformaldehyde, and then stained with H&E and Masson. Wound healing can be evaluated by the infiltration of inflammatory cells and the number of fibroblasts in the longitudinal section of the wound tissue under a microscope.

4.Experimental results

The results showed that compared with the wounds in the Normal group, the healing speed of the model group was significantly slower (P<0.05), indicating that the animal model used in this experiment was successfully modeled and could effectively ensure the accuracy of the research. Compared with the model group, the healing speed of diabetic wounds in the EGF group and Na-1 group was significantly accelerated (P<0.05). The representative images of the wound from 0 to 14 days and the time course curve of the healing rate are shown in Figure 1 and Figure 2 respectively. On the 14th day after the wound was caused, the H&E pathological observation results are shown in Figure 3. Compared with the normal group, the inflammatory cell infiltration was obvious in the model group, there was slight inflammatory infiltration in the EGF group, and there was no obvious inflammatory infiltration in the Na-1 group. The formation of collagen plays a key role in the wound healing process. It can promote cell migration and serve as the basis for extracellular matrix deposition during the proliferation phase of wound healing. Masson staining can be applied to evaluate the formation of collagen, in which collagen is stained. is blue, while cell bodies, muscle, and keratin are stained red. As shown in Figure 3, the model group has fewer collagen fibers, a loose network structure, and the epidermis is in an ulcerated state; while in the Na-1 group, the collagen fiber structure and epidermis are intact, and collagen fibers are accumulated in an orderly manner, indicating that Na-1 has a positive effect on wound healing. effect, which is consistent with the efficacy results in Figures 1 and 2.

Claims (9)

1. The application of a polypeptide in the preparation of a drug for promoting diabetic wound healing, characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1. 2. The application of a polypeptide in the preparation of an external dressing for promoting diabetic wound healing, characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1. 3. The application of a polypeptide in the preparation of a composition for promoting diabetic wound healing, characterized in that the composition is a gel containing a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1, and the gel also includes a gel. One or more of Bohm, hydroxypropyl methylcellulose, and sodium hyaluronate. 4. The use of the polypeptide according to claim 3 in preparing a composition for promoting diabetic wound healing, characterized in that the gel further includes the polypeptide, a moisturizing agent, a penetration enhancer, a pH regulator and water. 5. Application of the polypeptide according to claim 4 in preparing a composition for promoting diabetic wound healing, characterized in that the moisturizing agent is glycerol. 6. Application of the polypeptide according to claim 4 in preparing a composition for promoting diabetic wound healing, characterized in that the penetration enhancer is azone. 7. Application of the polypeptide according to claim 4 in preparing a composition for promoting diabetic wound healing, characterized in that the pH regulator is sodium hydroxide. 8. The use of the polypeptide according to claim 4 in preparing a composition for promoting diabetic wound healing, characterized in that the gel also contains a preservative, and the preservative is selected from the group consisting of potassium sorbate and ethyl p-hydroxybenzoate. One or more of ester, propyl parahydroxybenzoate and sodium benzoate. 9. The use of the polypeptide according to any one of claims 3 to 8 in preparing a composition for promoting diabetic wound healing, characterized in that the preparation method steps of the composition include: Weigh 0.4051g carbomer and 0.1031g hydroxypropyl methylcellulose and slowly add it to 60mL of distilled water, stir while adding, until completely dissolved; weigh an appropriate amount of sodium hydroxide and add it to the solution, stir while adding, and adjust the pH. When the value is 10, a colorless and transparent carbomer hydroxypropyl methylcellulose gel is obtained; then add 5.2g glycerin and stir evenly to obtain a gel matrix; weigh 0.12496g of polypeptide, add 20mL of water to disperse, and obtain a polypeptide solution; Add the polypeptide solution to the gel matrix and stir evenly. Finally, add 1.1g of azone and make up the weight to 100g with distilled water. Stir evenly to obtain a polypeptide external gel.