CN119775381A - A royal jelly polypeptide CMKI-21 and its application - Google Patents
A royal jelly polypeptide CMKI-21 and its application Download PDFInfo
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- CN119775381A CN119775381A CN202510050422.8A CN202510050422A CN119775381A CN 119775381 A CN119775381 A CN 119775381A CN 202510050422 A CN202510050422 A CN 202510050422A CN 119775381 A CN119775381 A CN 119775381A
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Abstract
本发明提供了一种能促进伤口愈合和抗皮肤光老化的蜂王浆多肽CMKI‑21及其应用，属于生物医药技术领域。所述蜂王浆多肽氨基酸序列为KKELVREVGFEICFKDHHEKI，通过固相化学合成法获得。本发明的蜂王浆多肽CMKI‑21分子量小、制备方法简单易行、纯度高且具有良好的生物相容性，能显著促进HaCaT细胞的增殖和迁移，加快小鼠全层皮肤缺损伤口的愈合速率，减轻HaCaT细胞的光损伤，缓解紫外线（UVB）造成的皮肤光老化和晒伤。该蜂王浆多肽有潜力作为伤口愈合剂应用于临床各类皮肤创伤，也可用于抗皮肤光老化和晒伤修复的药物和化妆品中。
The present invention provides a royal jelly polypeptide CMKI‑21 that can promote wound healing and resist skin photoaging and its application, belonging to the field of biomedicine technology. The royal jelly polypeptide amino acid sequence is KKELVREVGFEICFKDHHEKI, which is obtained by solid phase chemical synthesis. The royal jelly polypeptide CMKI‑21 of the present invention has a small molecular weight, a simple and easy preparation method, high purity and good biocompatibility. It can significantly promote the proliferation and migration of HaCaT cells, accelerate the healing rate of full-thickness skin defects in mice, reduce the light damage of HaCaT cells, and alleviate skin photoaging and sunburn caused by ultraviolet rays (UVB). The royal jelly polypeptide has the potential to be used as a wound healing agent in various types of clinical skin trauma, and can also be used in drugs and cosmetics for anti-skin photoaging and sunburn repair.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a royal jelly polypeptide CMKI-21 and application thereof.
Background
Skin is exposed to some deleterious physical, chemical and biological environments every day, and when skin cells are unable to defend against these external stimuli, their integrity and homeostasis is no longer maintained. Skin damage and premature aging are the result of continued exposure to the deleterious environment. In order for skin cells to be able to more aggressively cope with these external stimuli, we need to provide the skin with additional nutrients to increase the cell activity. At present, substances have been found which treat skin wounds and prevent aging. For example, polyphenols not only reduce inflammation, promote wound healing, but also reduce photoaging of the skin caused by ultraviolet radiation. Lipids and fatty acids can prevent water loss in the skin, which is critical to maintaining the integrity of the epidermal barrier. The polypeptide has various pharmacological activities such as antibiosis, antioxidation, immunoregulation and the like, and has good specificity and stability, so that the research interest of the polypeptide in the aspect of skin injury treatment is aroused.
As the most predominant cell type constituting the epidermis, keratinocytes play an important role not only as structural cells but also in wound healing and anti-skin aging. Proliferation and migration of keratinocytes can accelerate wound healing rate and restore barrier function of epidermis. UVB is the main cause of sunburn in the skin and acts primarily on epidermal keratinocytes. The long-term ultraviolet radiation not only accelerates the aging and death of the keratinocytes, but also indirectly promotes the expression of fibroblast extracellular matrix metalloproteinase (MMP-1) in a paracrine mode by cytokines secreted and released by the aging keratinocytes, and finally leads the skin to become rough and loose.
Royal jelly is a milky white or light yellow pasty substance secreted by the head of a worker bee of 5-15 days old by the glands of the head of the worker bee and the glands of the upper jaw, contains rich proteins, peptides and free amino acids, has various biological activities of resisting bacteria, resisting inflammation, promoting wound healing, resisting oxidation and the like, and is a natural health-care food. Researchers have found that royal jelly polypeptides have more desirable pharmacological functions than royal jelly proteins and are easier to store. At present, less researches are carried out on the royal jelly polypeptide for promoting wound healing and resisting skin photoaging, so that the research on the royal jelly polypeptide capable of promoting wound healing, preventing and repairing skin ultraviolet injury has important clinical significance.
Disclosure of Invention
The invention aims to provide an application of a royal jelly polypeptide as a functional ingredient in the aspects of promoting wound healing and resisting skin photoaging.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The invention provides a royal jelly polypeptide CMKI-21, the amino acid sequence of the royal jelly polypeptide CMKI-21 is Lys-Lys-Glu-Leu-Val-Arg-Glu-Val-Gly-Phe-Glu-Ile-Cys-Phe-Lys-Asp-His-His-Glu-Lys-Ile（KKELVREVGFEICFKDHHEKI）,, the molecular weight is 2584.8 Da, and the structure is as follows:
The invention also provides an application of the royal jelly polypeptide CMKI-21 in promoting proliferation and migration of skin cells.
Further, the skin cells are human immortalized keratinocytes, and the concentration of the royal jelly polypeptide CMKI-21 is 10 -11~10-6 M.
The invention also provides application of the royal jelly polypeptide CMKI-21 in preparation of a preparation for promoting skin wound healing.
Further, the skin wounds include wounds resulting from physical and chemical injury.
Further, the preparation for promoting skin wound healing further comprises pharmaceutically acceptable auxiliary materials.
The invention also provides application of the royal jelly polypeptide CMKI-21 in preparing cosmetics for resisting skin photoaging or sunburn repair.
Further, the cosmetic comprises spray, gel, jelly, emulsion and water agent, and the concentration of the royal jelly polypeptide CMKI-21 in the cosmetic is 10 -9~10-5 M.
The invention has the beneficial effects that:
the polypeptide of the invention is derived from royal jelly protein, can promote proliferation and migration of human immortalized keratinocytes (HaCaT), and has no cytotoxicity. Experiments prove that the polypeptide can obviously inhibit the apoptosis of keratinocytes damaged by ultraviolet induction, promote the wound repair of full-layer skin damage of mice and reduce the photo-aging of the skin of the mice caused by ultraviolet irradiation. Therefore, the royal jelly polypeptide CMKI-21 has good application prospect in the field of medicines and cosmetics.
Drawings
FIG. 1 shows a high performance liquid chromatogram of royal jelly polypeptide CMKI-21.
FIG. 2 shows a mass spectrum of royal jelly polypeptide CMKI-21.
FIG. 3 is a graph showing the effect of Lac Regis Apis polypeptide CMKI-21 on activity of HaCaT cells, wherein Control represents Control wells, 10 -12、10-11、10-10、10-9、10-8、10-7、10-6、10-5 M represents intervention wells for each concentration of Lac Regis Apis polypeptide, (A) is a graph showing cell activity of HaCaT cells treated with Lac Regis Apis polypeptide CMKI-21 at different concentrations 48 h, and (B) is a graph showing cell activity of HaCaT cells treated with Lac Regis Apis polypeptide CMKI-21 at different concentrations 72 h, wherein p <0.05, and p <0.01.
Fig. 4 is a graph showing the effect of royal jelly polypeptide CMKI-21 on the scratch healing ability of HaCaT cells, wherein Control represents a Control well, 10 -11、10-10、10-9、10-8 M represents an intervention well of royal jelly polypeptide at each concentration, a microscopic graph of cell scratch healing at different time points after adding royal jelly polypeptide at different concentrations into a scratch model of HaCaT cells, and a graph of analysis of scratch healing rate after HaCaT cells are treated with different concentrations CMKI-21 for a certain period of time, wherein p <0.05 and p <0.001.
FIG. 5 is a graph showing the effect of Lac Regis Apis polypeptide CMKI-21 on HaCaT cell migration ability, wherein Control represents Control wells, 10 -11、10-10、10-9、10-8 M represents Lac Regis Apis polypeptide intervention wells of various concentrations, (A) is a microscopic image of HaCaT cell migration after adding different concentrations of Lac Regis Apis polypeptide into a Transwell lower chamber, 24h, and (B) is a graph of cell mobility analysis after 24h of HaCaT cells treated with CMKI-21, wherein p <0.05, p <0.01.
Fig. 6 is a graph showing the effect of royal jelly polypeptide CMKI-21 on the activity of HaCaT cells irradiated with UVB (30 mj/cm 2 x 3), wherein Control represents Control, 10 -11、10-10、10-9、10-8 M represents the concentration of royal jelly polypeptide in the intervening wells, model represents Model, and p <0.05, p <0.0001.
Fig. 7 is a graph showing the effect of royal jelly polypeptide CMKI-21 on the wound of the full-thickness skin defect of a mouse, wherein Control represents a Control group, EGF (10 -7 M) represents a positive Control group, 10 -10、10-9、10-8 M represents an intervention group of the royal jelly polypeptide at each concentration, wherein (A) is a graph for macroscopic evaluation of the wound of the skin of the mouse after the treatment of the royal jelly polypeptide CMKI-21, and (B) is a statistical graph for the wound healing rate of the full-thickness skin defect of the mouse after the treatment of the royal jelly polypeptide CMKI-21, wherein p <0.05 and p <0.01.
FIG. 8 shows the effect of royal jelly polypeptide CMKI-21 on repairing photoaging of mouse skin, wherein Control represents Control group, model represents Model group, and 10 -8、10-7、10-6、10-5 M represents intervention group of royal jelly polypeptide of each concentration.
Detailed Description
In order to make the content of the invention more easily understood, the technical scheme of the invention is further described below with reference to specific embodiments. The examples are intended to illustrate the invention, but not to limit it in any way.
EXAMPLE 1 solid-phase chemical Synthesis of Lac Regis Apis polypeptide CMKI-21
The amino acid sequence of the royal jelly polypeptide CMKI-21 is KKELVREVGFEICFKDHHEKI (SEQ ID NO. 1) and the molecular weight is 2584.8 Da. Royal jelly polypeptide CMKI-21 is synthesized by Jiangsu Style biotechnology Co.
The high performance liquid chromatography separation result is shown in figure 1, and in the high performance liquid chromatography analysis, a single peak appears at 7.725 min parts of the royal jelly protein sample, and the area ratio reaches more than 98%, which indicates that the purity of the synthesized polypeptide meets the requirement. Therefore, the component corresponding to the peak was selected for mass spectrometry, and the molecular weight thereof was verified by mass spectrometry.
The mass spectrum analysis results are shown in fig. 2, and the mass spectrum analysis shows that under the positive ion mode, the first-order mass spectrum of CMKI-21 has ion peaks with different valence states, M/z=517.9 ([ M+5H ] 5+）、m/z＝627.2（[M+4H]4+) and the like, and according to a molecular weight deduction formula, the molecular weight= (M/s value multiplied by charge number-charge number multiplied by proton mass), the calculated molecular weights are 2584.8 Da, and the molecular weight accuracy of CMKI-21 is proved.
Example 2 Effect of Royal jelly polypeptide CMKI-21 on HaCaT cell Activity
Human immortalized keratinocytes (HaCaT) were prepared as a 3.5x10 4 cells/mL cell suspension in DMEM medium (10% fetal bovine serum, 1% penicillin-streptomycin) and plated in 96-well plates at 100 μl per well. After culturing 24h, starving 12. 12 h with serum-free medium, dissolving the Lac Regis Apis polypeptide CMKI-21 in serum-free medium, adding 100 μl of Lac Regis Apis polypeptide CMKI-21（0、10-12、10-11、10-10、10-9、10-8、10-7、10-6、10-5M）, per well, and culturing in CO 2 incubator. After treatment with royal jelly polypeptide CMKI-21 of 48 h and 72 h, respectively, 10. Mu.L of MTT was added to each well, incubated in a CO 2 incubator in the absence of light for 4h, the supernatant was discarded, 100. Mu.L of dimethyl sulfoxide (DMSO) was added to each well, and shaking was performed at room temperature for 15 minutes, and absorbance was measured at 490 nm using an enzyme-labeled instrument.
Cell activity was calculated from cell activity = OD Test group /OD Control group x 100%.
As shown in FIG. 3, compared with the Control group, the proliferation of HaCaT cells can be remarkably promoted after the action of the royal jelly polypeptide CMKI-21 of 10 -9、10-8 and 10 -6 M is 48 h, and the proliferation of HaCaT cells can be remarkably promoted after the action of the royal jelly polypeptide CMKI-21 of 10 -11、10-10、10-9、10-8 and 10 -7 M is 72 h.
Example 3 healing-promoting Activity of Royal jelly polypeptide CMKI-21 on HaCaT cell scratch model
Inserts (ibidi) were placed in 24 well cell culture plates, 70 μl of HaCaT cell suspension (3.5×10 4 cells/chamber) was inoculated into each insert chamber, the inserts were removed after culturing 12. 12 h to form regular scratch areas, then 1 mL royal jelly polypeptides (0, 10 -11、10-10、10-9、10-8 M) were added, and the scratch areas were microscopically recorded at 0, 12 and 24 h treated with royal jelly polypeptides CMKI-21, respectively.
As shown in FIG. 4, the cell scratch area can be remarkably reduced after the royal jelly polypeptides CMKI-21 of 10 -11、10-10 and 10 -9 M act on 24 h.
EXAMPLE 4 Effect of Royal jelly polypeptide CMKI-21 on HaCaT cell migration
1X 10 5 HaCaT cells were inoculated into each Transwell cell, 600. Mu.L of royal jelly polypeptide CMKI-21 (0, 10 -11、10-10、10-9、10-8 M) was added into the lower cell, and cultured in a CO 2 incubator for 24 h. Cells were fixed with paraformaldehyde 20 min followed by staining with 0.1% crystal violet solution 20 min. PBS (ph=7.4) was washed twice, the cells not migrated in the upper chamber were gently rubbed with a cotton swab, the bottom of the chamber was observed with a microscope, and photographed.
As shown in FIG. 5, the royal jelly polypeptides CMKI-21 of 10 -11 and 10 -10 M significantly promote migration of HaCaT cells.
Example 5 Effect of Lac Regis Apis polypeptide CMKI-21 on UVB-irradiated HaCaT cell Activity
HaCaT cells were prepared as a cell suspension of 4X 10 3 cells/mL in DMEM medium (containing 10% fetal calf serum, 1% penicillin-streptomycin) and plated in 96-well plates, 100. Mu.L per well, and incubated in a CO 2 incubator for 24 h. The dosing group (10 -11、10-10、10-9、10-8) was pretreated with serum-free medium containing the royal jelly polypeptide CMKI-21 (10 -11、10-10、10-9、10-8 M) 12h, and the Model group (Model) and the Control group (Control) were cultured with serum-free medium containing no royal jelly polypeptide. 12 After h, the medium was removed and washed twice with PBS (ph=7.4) buffer leaving 35 μ LPBS (ph=7.4) buffer per well. The wells of the control group were covered with tinfoil and the model and dosing groups were exposed to 30 mj/cm 2 of ultraviolet radiation by placing the 96-well plates under UVB (313 nm) lamp. Irradiation was performed 2 times daily, at intervals of 12h, and 3 times continuously. After each irradiation, the PBS buffer solution (pH=7.4) was discarded from each well, 100. Mu.L of serum-free medium containing the royal jelly polypeptide CMKI-21 (10 -11、10-10、10-9、10-8 M) was added to the dosing group, the model group and the control group were recovered with serum-free medium containing no royal jelly polypeptide, and the cell activity was measured with CCK8 kit after the third irradiation of 24. 24 h.
The results are shown in fig. 6, where the HaCaT cell activity of the model group was significantly reduced compared to the control group, indicating that UVB irradiation caused severe damage to the cells. Compared with the model group, the royal jelly polypeptides CMKI-21 of 10 -10 and 10 -9 M can obviously improve the activity of HaCaT cells.
EXAMPLE 6 Effect of Royal jelly polypeptide CMKI-21 on full skin Defect wounds in mice
After 35 female Kunming mice were acclimatized for 1 week for 5-6 weeks, back hair was removed with an electric razor, and then the depilatory cream was applied to the exposed skin, and 10min were then rubbed with gauze to completely remove back hair. The following day, mice were anesthetized with 5% (w/v) sultai solution (50 mg/kg) under sterile conditions. Two circular wounds with the diameter of about 6mm are manufactured on the back of the mouse by using a puncher, photographing is carried out after the wounds are dried, and the area of the skin wound of the mouse is calculated by using Image J software, namely the initial wound area.
After the full-thickness skin defect wound model of the mice is established successfully, the mice are randomly divided into 5 groups of 7 mice each. Control (blank), EGF (10 -7 M, positive Control) and Lac Regis Apis polypeptide CMKI-21 (10 -10、10-9、10-8 M), respectively. Wherein the Control group was coated with 20. Mu.L of PBS (pH=7.4) buffer, the positive Control group was coated with 20. Mu.L of epidermal growth factor (EGF, 10 -7 M), and the royal jelly polypeptide group was coated with 20. Mu.L of PBS buffer containing CMKI-21 (10 -10、10-9 and 10 -8 M).
The method comprises the following steps of observing wound change every day, photographing wounds of mice on days 1,3,5 and 7, and calculating the wound area by adopting Image J software, namely the unhealed wound area, wherein the calculation formula is as follows:
As shown in FIG. 7, the wound healing rate of mice treated with the royal jelly polypeptide CMKI-21 was significantly improved in the group of royal jelly polypeptides (10 -9、10-8 M) on days 5 and 7.
EXAMPLE 7 repair of the light aging of the mouse skin by the Lac Regis Apis polypeptide CMKI-21
30 Female Kunming mice, 5-6 weeks old, were randomly assigned to 6 groups after 1 week of adaptive rearing, 5 each, i.e., control group, model group and royal jelly polypeptide CMKI-21 group (10 -8、10-7、10-6、10-5 M). Removing back hair with an electric shaver before ultraviolet irradiation, further applying the depilatory cream on the exposed skin, and wiping with gauze after 10min to completely remove back hair. The Control group was not irradiated with ultraviolet radiation, and the Model group and the royal jelly polypeptide CMKI-21 group were irradiated with UVB (313, nm) at intervals of 3 times a week, the single irradiation dose of the first week was 60 mj/cm 2, and then the single irradiation dose per week was increased by 30 mj/cm 2 for four weeks. After uv irradiation, the Model group mice were uniformly coated with 200 μl of PBS (ph=7.4) buffer on the back, and the royal jelly polypeptide CMKI-21 groups were uniformly coated with 200 μl of PBS buffer containing royal jelly polypeptide CMKI-21 (concentration 10 -8、10-7、10-6、10-5 M) on the back, respectively, 1 time a day. The mice were observed daily for skin changes, newly grown mouse hairs were removed, and each group of mice was evaluated for skin damage at 24 h after the 28 th application of active polypeptide.
As a result, as shown in FIG. 8, the skin of the Control group mice was firm, smooth, wrinkle-free, and showed a healthy skin color. The Model group mice had reddish skin, became dry, desquamated, and had marked sunburn, wrinkles, and hyperkeratosis. Royal jelly polypeptide CMKI-21 (10 -7、10-6、10-5 M) has slightly red skin color, and can obviously improve photodamage characteristics of sunburn, dryness, desquamation, etc. compared with Model group.
In summary, the present invention provides a royal jelly polypeptide CMKI-21 having a wound healing promoting activity and an anti-skin photoaging activity. The polypeptide is easy to synthesize and has good biocompatibility, and has good application prospect in the aspects of skin wound treatment and sunburn repair.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (8)
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