A recombinant type IV collagen composition for repairing basement membrane and soothing and reducing redness

Abstract

The present invention provides a recombinant type IV collagen composition for repairing basement membrane and soothing and reducing redness. The inventors found that the recombinant type IV collagen described in the specification of this patent inhibits the phosphorylation of VE-cadherin protein by mediating the PKCα / PYK2 / Src signaling pathway, protects the tight junctions between endothelial cells, prevents the leakage of blood components, and reduces vascular permeability; it can also improve the dissociation of the VE-cadherin complex caused by oxidative stress and maintain the homeostasis of the endothelial barrier; therefore, it has good effects of repairing the basement membrane barrier and soothing and reducing redness. The inventors also first discovered that the repair peptide (GAAGLPGPK) has the effects of repairing the basement membrane barrier, soothing and reducing redness, and the combination of a certain proportion of the recombinant type IV collagen and the repair peptide shows synergistic effects in repairing the basement barrier and soothing and reducing redness.

Description

Technical Field

[0001] The present invention relates to the technical field of efficacy cosmetics or topical drugs, and specifically relates to recombinant type IV collagen and repair functional peptides with the functions of repairing basement membrane barrier and soothing and reducing redness, and their applications in the preparation of cosmetics or topical drugs with the functions of repairing basement membrane barrier and soothing and reducing redness. Background Art

[0002] Collagen, as an important structural protein, is widely present in animal tissues. Type IV collagen is one of the main components of the basement membrane and plays an important role in maintaining the structure and function of tissues. Traditional type IV collagen is mainly extracted from animal tissues, which has problems such as limited sources, possible pathogen carriage, and large differences between batches.

[0003] In the field of skin health and repair, the basement membrane barrier plays a crucial role. It not only maintains the structural integrity of the skin but also participates in the regulation of numerous physiological functions. However, in daily life, the skin is frequently invaded by external harmful factors, such as ultraviolet (UVA and UVB) irradiation, which can damage the basement membrane barrier, resulting in a significant decrease in the content of important basement membrane proteins such as laminin, and further disrupting the normal physiological functions of the skin, leading to a series of skin problems, such as skin relaxation, wrinkle formation, and accelerated water loss.

[0004] Meanwhile, in the in vivo environment, the endothelial barrier also faces many challenges. Reactive oxygen species such as hydrogen peroxide (H2O2), which can be generated by the body's metabolic processes or external stimuli, can damage the integrity of endothelial cell adhesion junctions, specifically manifested as the dissociation of the VE-cadherin complex, leading to impaired endothelial barrier function. This damage interferes with the tight junctions between cells, resulting in abnormal substance permeation and affecting the normal internal environment homeostasis of tissues, which is closely related to the occurrence and development of various inflammatory and vascular-related diseases.

[0005] In addition, the number of people with sensitive skin is increasing day by day, and the skin is prone to discomfort symptoms such as redness and stinging, which not only affect the appearance but also reduce the quality of life. Although there are some countermeasures in the prior art for these skin problems, there is still a lack of efficient and highly targeted solutions in terms of repairing the basement membrane barrier, protecting the endothelial barrier, and soothing sensitive skin, and it is difficult to meet the urgent needs of people for skin health maintenance and repair. Summary of the Invention

[0006] In view of the above technical problems existing in the prior art, the purpose of the present invention is to provide a recombinant type IV collagen with good functions of repairing basement barrier and soothing and reducing redness, small peptides that synergistically enhance the effect with the recombinant type IV collagen, and their uses in the preparation of cosmetics or topical drugs for repairing basement barrier and soothing and reducing redness.

[0007] The inventors conducted in-depth research to solve the above technical problems and found that: 1. The recombinant type IV collagen described in the patent specification has good effects of repairing the basal layer and soothing and reducing redness; 2. For the first time, a nonapeptide (GAAGLPGPK) with the effects of repairing the basement membrane barrier and soothing and reducing redness was discovered and named the repair nonapeptide; 3. It was further found that a combination of the recombinant type IV collagen and the nonapeptide in a certain proportion showed synergistic effects in repairing the basal barrier and soothing and reducing redness, thus completing the present invention.

[0008] That is, the technical solution of the present invention includes:

[0009] 1. Use of a recombinant type IV collagen in the preparation of a cosmetic or a topical medicament for repairing the basal barrier and soothing and reducing redness; wherein, the recombinant type IV collagen is used as the ingredient for repairing the basal barrier and soothing and reducing redness of the cosmetic or the topical medicament, and its amino acid sequence is as shown in SEQ ID No:1.

[0010] SEQ ID No:1

[0011] GPPGPPGIVIGTGPLGEKGERGYPGTPGPRGEPGPKGFPGLPGQPGPPGLPVPGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLPGPPGSPGPPGQPGYTNGIVECQPGPPGDQGPPGIPGQPGFIGEIGEKGQKGESCLICDIDGYRGPPGPQGPPGEIGFPGQPGAKGDRGLPGRDGVAGVPGPQGTPGLIGQPGAKGEPGEFYFDLRLKGDKGDPGFPGQPGMPGRAGSPGRDGHPGLPGPKGSPGSVGLKGERGPPGGVGFPGSRGDTGPPGPP

[0012] 2. The use according to item 1, wherein the cosmetic or the topical medicament is prepared in a form suitable for application to the skin.

[0013] 3. The use according to item 1, wherein the cosmetic is selected from lotion, essence, cream or mask.

[0014] 4. A polypeptide having the effects of repairing the basal barrier and soothing and reducing redness, and its amino acid sequence is as shown in SEQ ID No:2.

[0015] SEQ ID No:2

[0016] GAAGLPGPK

[0017] 5. A recombinant collagen composition with the effects of repairing the basal barrier and soothing and reducing redness, which contains recombinant type IV collagen and repairing nonapeptide as active ingredients; wherein, in the recombinant collagen composition, the weight ratio of the recombinant type IV collagen to the repairing nonapeptide is 500:1 to 10:1;

[0018] The amino acid sequence of the recombinant type IV collagen is as shown in SEQ ID No:1;

[0019] The amino acid sequence of the repairing nonapeptide is as shown in SEQ ID No:2.

[0020] 6. The recombinant collagen composition according to item 5, wherein the weight ratio of the recombinant type IV collagen to the repairing nonapeptide is 50:1 to 100:1.

[0021] 7. A cosmetic or external medicine with the effects of repairing the basal barrier and soothing and reducing redness, which contains the recombinant collagen composition described in item 5.

[0022] 8. The cosmetic or external medicine according to item 7, wherein the cosmetic or external medicine is prepared in a form suitable for application to the skin.

[0023] 9. The cosmetic according to item 8, wherein the cosmetic is selected from lotion, essence, cream or mask.

[0024] 10. Use of the recombinant collagen composition according to item 4 in the preparation of a cosmetic or external medicine for repairing the basal barrier and soothing and reducing redness; wherein the recombinant type IV collagen and the repairing nonapeptide are used as the ingredients for repairing the basal barrier and soothing and reducing redness of the cosmetic or external medicine.

[0025] 11. The use according to item 10, wherein the cosmetic or external medicine is prepared in a form suitable for application to the skin.

[0026] 12. The use according to item 10, wherein the cosmetic is selected from lotion, essence, cream or mask.

[0027] Inventive effects

[0028] Both the recombinant type IV collagen and the repairing nonapeptide have obvious biological activities of repairing the basal barrier and soothing and reducing redness, and when combined in a certain proportion, they show synergistic effects in repairing the basal barrier and soothing and reducing redness.

[0029] The experimental results of mechanism research show that the recombinant type IV collagen can repair the basement membrane barrier damage caused by ultraviolet rays; it can inhibit the dissociation of the VE-cadherin / p120-catenin / β-catenin protein complex, effectively maintain the stability of the inter-endothelial cell junction protein complex, and protect the endothelial barrier function. The results of human efficacy evaluation show that the recombinant type IV collagen has a significant effect on improving skin flushing. Description of the Drawings

[0030] Figure 1 Immunofluorescence photographs showing the amount of laminin in each group.

[0031] Figure 2 Graph showing the changes in the proteins of the PKCα / Src / PYK2 / VE-cadherin signaling pathway under the intervention of different concentrations of recombinant type IV collagen detected by Western Blot.

[0032] Figure 3 Graph showing the changes in the proteins of the VE-cadherin, p120-catenin, and β-catenin complexes under the intervention of different concentrations of recombinant type IV collagen detected by immunoprecipitation.

[0033] Figure 4 Graph showing the changes in the EI value of skin heme at different time points.

[0034] * indicates a significant difference compared with the Do group, ***P < 0.001

[0035] Figure 5 Graph showing the changes in the a* value of skin color (red - green) at different time points.

[0036] * indicates a significant difference compared with the Do group, ***P < 0.001

[0037] Figure 6 Graph showing the changes in the red area at different time points.

[0038] * indicates a significant difference compared with the Do group, **P < 0.01, ***P < 0.001 Detailed Implementation Modes

[0039] Example 1 Preparation of Recombinant Type IV Collagen and Repairing Nonapeptide

[0040] Referring to Examples 1 and 2 of the specification of Chinese Patent Application Publication CN117903287A, the recombinant type IV collagen was prepared and purified.

[0041] The repairing nonapeptide was commissioned to be synthesized by Jier Biochemical (Shanghai) Co., Ltd. The polypeptide sequence is GAAGLPGPK, and its amino acid sequence and purity were confirmed by mass spectrometry and high-performance liquid chromatography.

[0042] Example 2: Effects of recombinant type IV collagen, repairing nonapeptides and their combination on repairing basement membrane barrier

[0043] The following samples were prepared: recombinant type IV collagen (concentration of 1 mg / mL, aqueous solution), repair nonapeptides 1-5 (concentrations of 0.001 mg / mL, 0.002 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.1 mg / mL, respectively, aqueous solution), and compositions 1-5 (recombinant type IV collagen at 0.2 mg / mL + repair nonapeptide concentrations of 0.001 mg / mL, 0.002 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.1 mg / mL, respectively, aqueous solution).

[0044] Positive control: 100 μg / mL vitamin C + 7 μg / mL vitamin E

[0045] Irradiation and sample intervention: Hacat cells were plated in 6-well plates (5*10 4 / well), 2 mL was added to each well and cultured in a 37°C, 5% CO2 incubator. After 24 hours of culture, irradiation and sample intervention were started. A normal group, a model group, a positive control group, and each sample group were set up with 3 parallel wells in each group. The modeling method was UV modeling, and the irradiation dose was UVA (30 J / cm 2 ) and UVB (75mJ / cm 2 After irradiation, the model group received fresh culture medium, while the positive control and sample groups received fresh culture medium containing samples for intervention. A volume of 2 mL was added to each well. The normal group only received culture medium replacement without irradiation or sample intervention.

[0046] Laminin was detected by immunofluorescence: after washing with PBS, the cells were fixed with 4% paraformaldehyde for 15 minutes before immunofluorescence detection. The cells were observed under a microscope, and the images were collected and analyzed. Figure 1 In the figure, the intensity of red fluorescence represents the amount of laminin.

[0047] Calculate the laminin recovery rate of the positive control group or each sample group. At the same time, calculate the combination index CI of each sample group = (A×B) / AB. When CI < 1, it indicates that the combination has a synergistic effect; when CI ≤ 0.8, it indicates that the synergistic effect is very significant.

[0048] C = laminin in the normal group minus laminin in the model control group, representing the upper limit of laminin recovery after sample intervention;

[0049] T = The laminin amount in each sample group - The laminin amount in the model control group, representing the actual level of laminin amount restored after administration of each sample;

[0050] T / C represents the laminin recovery rate of the positive control group or each sample group;

[0051] A and B respectively represent the (1 + T / C) values when recombinant type IV collagen or repair nonapeptide is administered alone;

[0052] AB represents the (1 + T / C) value when the composition 1 - 5 is administered.

[0053] Experimental conclusion:

[0054] The laminin recovery rates and CI values of the positive control group and each sample group are shown in Table 1.

[0055] Table 1 Immunofluorescence detection of laminin expression level (n = 3)

[0056]

[0057] Compared with the normal group, the laminin content in the model group decreased significantly, indicating that the stimulation conditions in this experiment were effective and the modeling was successful. Compared with the model group, the laminin content in each sample group increased significantly, indicating that each sample had a significant effect on repairing the basement membrane barrier. The combination drug indices of the above compositions 2, 3, 4, and 5 were all less than 1, indicating that there was a synergistic effect when the weight ratio of recombinant type IV collagen to repair nonapeptide was 500 - 10:1. Moreover, the synergy indices of compositions 3 and 4 were less than 0.9, indicating that when the ratio of recombinant type IV collagen to repair nonapeptide was 100 - 50:1, the synergistic effect between the two was very significant. Among them, the synergistic effect of composition 3 was the most significant.

[0058] This can illustrate that recombinant type IV collagen, repair nonapeptide and their compositions can promote the expression of important proteins in the basement membrane after injury, and have the effects of repairing the basement membrane barrier and soothing and reducing redness.

[0059] Example 3: Study on the maintenance of the homeostasis of endothelial cell adhesion junction-related proteins by recombinant type IV collagen

[0060] Immunoblot analysis:

[0061] HUVEC cells were seeded in 100 mm cell culture dishes at a seeding density of 1.2×106 cells / dish and cultured overnight until the cell confluence reached 60% - 70%. Cells were then treated with recombinant type IV collagen at different concentrations (0.025, 0.05, and 0.1 mg / mL), and a normal group (C) and a model group (M) were set up. After 24 hours, according to the manufacturer's instructions of the total protein extraction kit (BC3711, Solarbio, China), HUVEC cells were lysed to obtain protein samples. Equal amounts of protein samples were separated on 7.5% SDS-PAGE gels and transferred to 0.45 μM PVDF membranes using different currents according to the size of the protein molecules. The membranes were further blocked with 5% non-fat milk. Then the membranes were incubated with primary antibodies including β-actin, PKCα, phosphorylated PKCα, Src, phosphorylated Src, PYK2, phosphorylated PYK2, VE-cadherin, and phosphorylated VE-cadherin at 4°C for 16 hours. Horseradish peroxidase-conjugated affinity-pure goat anti-rabbit IgG (H+L) (SA00001-2, ) was used to detect the antigen-antibody complexes. Finally, an enhanced chemiluminescence kit was used for color development, and the immunoreactive bands were quantitatively analyzed by densitometric scanning using ImageJ software. This experiment can detect the expression levels of various proteins and their phosphorylation states under different treatment conditions, which helps to analyze the protein expression and the activation of signal pathways.

[0062] Co-immunoprecipitation analysis:

[0063] First, HUVEC cells were lysed, and an appropriate amount of IP cell lysis buffer and protease inhibitor were added to collect the supernatant. A small amount of the supernatant was used as the input sample (Input) in the co-immunoprecipitation (Co-IP) experiment. In the negative control (IgG) group, 1.0 μg of normal IgG of the same species as the precipitation antibody used in the IP experiment and 20 μL of protein A / G magnetic beads (thoroughly mixed before use) were added to the protein supernatant; in the experimental group, 20 μL of protein A / G magnetic beads were directly added to the supernatant, and the mixture was incubated with shaking at 4 °C for 1 hour. The remaining supernatant was taken out and placed in a new tube, and it was continuously stirred and incubated with VE-cadherin antibody at 4 °C for 2 hours. Then, protein A / G magnetic beads were added to the supernatant / antibody mixture, and the mixture was continuously stirred and incubated overnight at 4 °C. The magnetic beads were washed three times with ice-cold IP lysis buffer. The bound proteins were eluted with 1× protein sample buffer and subjected to SDS-PAGE electrophoresis. Finally, detection was performed by Western Blot experiment using VE-cadherin, p120-catenin, and β-catenin antibodies. This experiment can be used to study the interaction between proteins, and here it can be used to detect the binding state between VE-cadherin and catenin.

[0064] Experimental conclusion:

[0065] Table 2 Quantitative analysis table of immunoblot detection

[0066]

[0067] * indicates significant difference compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001

[0068] Western Blot was used to detect the effects of recombinant type IV collagen on PKCα / Src / PYK2 / VE-cadhrein and their phosphorylated proteins. The protein electrophoresis diagram is as Figure 2 shown. The normal group was not affected by type IV collagen. The protein bands were analyzed by ImageJ software, and the data were analyzed by EXCEL to calculate STDEV and T-test. Compared with the model group, recombinant type IV collagen significantly reduced the p-PKCα / PKCα, p-Src / Src, p-PYK2 / PYK2, and p-VE-cadherin / VE-cadherin ratios in a dose-dependent manner. These data indicate that recombinant type IV collagen reduces the level of phosphorylated VE-cadherin by regulating PKCα / Src / PYK2.

[0069] To confirm whether VE-cadherin phosphorylation disrupts the β-catenin–p120-catenin–VE-cadherin complex, HUVEC cells were treated with 500 μM H2O2 for 4 hours, VE-cadherin was immunoprecipitated, and then the presence of p120-catenin and β-catenin in the immunoprecipitate was evaluated( Figure 3 ). There was no significant difference in VE-cadherin expression among the groups. The levels of p120-catenin and β-catenin in the model group were significantly decreased. Treatment of cells with recombinant type IV collagen for 24 hours before modeling detected a dose-dependent increase in p120-catenin and β-catenin. Recombinant type IV collagen inhibited the dissociation of β-catenin and p120-catenin from the VE-cadherin complex induced by H2O2. These results indicate that H2O2 disrupts the integrity of endothelial cell adhesion junctions, leading to the disassembly of the VE-cadherin complex, while recombinant type IV collagen can protect the VE-cadherin complex, maintain the interaction between adhesion junction-related proteins, strengthen the connection between endothelial cells, and thus protect the integrity of the endothelial barrier.

[0070] Overall, the results of immunoprecipitation and Western Blot corroborate each other, elaborating in detail the mechanism of H2O2-induced disruption of endothelial barrier-related proteins and complexes at the molecular level, as well as the protective mechanism of recombinant type IV collagen, providing a solid basis for a deeper understanding of the cellular biological basis behind skin soothing and redness reduction problems and the development of targeted treatment strategies.

[0071] Example 4: Evaluation of the soothing and redness-reducing human efficacy of recombinant type IV collagen and Composition 3

[0072] Before the experiment, a lactic acid sting test was conducted on the subjects, and 30 subjects with sensitive skin were selected for facial evaluation and randomly divided into the recombinant type IV collagen and Composition 3 groups. The hemoglobin content (EI) was measured using a narrow-band reflectance spectrophotometer (Mexameter MX18). The redness area and skin color (red-green) value (a* value) were measured and analyzed using a high-resolution camera and Image-Pro Plus software. Once in the morning and once in the evening every day, recombinant type IV collagen or Composition 3 (sample group) was applied to the left cheek, and a matrix without recombinant type IV collagen or Composition 3 (control group) was applied to the right cheek. The EI value, redness area, and a* value were measured at day 0 (D0) before treatment, day 14 (D14) after treatment, and day 28 (D28) after treatment.

[0073] Experimental conclusion:

[0074] The skin hemoglobin content (EI) values of 30 subjects were obtained. After treatment with the recombinant type IV collagen (concentration: 1 mg / mL, aqueous solution) or Composition 3 prepared in Example 2 for 14 days and 28 days, there were significant differences in the EI values. Similarly, after using Matrix 1 without recombinant type IV collagen or Composition 3 for 14 days and 28 days (control group), there were also significant differences in the EI values. After 14 days of use, the EI value in the recombinant type IV collagen group decreased by 11.22%, the EI value in the Composition 3 group decreased by 21.97%, and the EI value in the control group decreased by 3.80%, with significant differences among the groups. After 28 days of use, the EI value in the recombinant type IV collagen group decreased by 20.19%, the EI value in the Composition 3 group decreased by 32.17%, and the EI value in the control group decreased by 8.23%, with significant differences among the groups( Figure 4 ). These findings indicate that both recombinant type IV collagen and Composition 3 have a soothing effect after 14 days and 28 days of use. The soothing effects of the recombinant type IV collagen group and the Composition 3 group are better than those of the control group at both time points, and the effect of the Composition 3 group is better than that of the recombinant type IV collagen group.

[0075] In addition, there were significant differences in the skin color (red - green) values (a* values) of 30 subjects after 14 days and 28 days of using the recombinant type IV collagen or Composition 3 (sample group), while there were no significant differences in the a* values after using Matrix 1 without recombinant type IV collagen or Composition 3 (control group) for 14 days and 28 days. After 14 days, the a* value in the recombinant type IV collagen group decreased by 11.56%, the Composition 3 group decreased by 19.76%, and the control group decreased by 3.86%, with significant differences among the groups; after 28 days, the a* value in the recombinant type IV collagen group decreased by 9.15%, the Composition 3 group decreased by 18.35%, and the control group decreased by 1.64%, with significant differences among the groups( Figure 5 ) This further demonstrates that both recombinant type IV collagen and Composition 3 have a soothing effect after 14 days and 28 days of use. The soothing effects of the recombinant type IV collagen group and the Composition 3 group are better than those of the control group at both time points, and the effect of the Composition 3 group is better than that of the recombinant type IV collagen group.

[0076] In addition, compared with the red - swollen area before use, there were significant differences in the red - swollen area of 30 subjects after 14 days and 28 days of using the recombinant type IV collagen or Composition 3 (sample group), while there were no significant differences in the red - swollen area after using Matrix 1 without recombinant type IV collagen or Composition 3 (control group) for 14 days and 28 days. After 14 days of using the recombinant type IV collagen, the red - swollen area in the sample group decreased by 10.55%, the Composition 3 group decreased by 14.98%, and the control group decreased by 0.11%, with significant differences between the two groups. After 28 days, the red - swollen area in the recombinant type IV collagen group decreased by 9.75%, the Composition 3 group decreased by 16.04%, and the control group decreased by 1.92%, with significant differences between the two groups(Figure 6 )。These results further indicate that recombinant type IV collagen or Composition 3 has a soothing effect both at 14 days and 28 days of use, and the soothing effect of the sample group is better than that of the control group at both time points.

[0077] Combined with the above data, it can be concluded that recombinant type IV collagen and Composition 3 can significantly reduce redness and soothe the skin after 14 days and 28 days of use.

Claims

1. A recombinant type IV collagen with the activity of repairing basal barrier and soothing redness, the amino acid sequence of which is shown in SEQ ID No:

1.

2. Use of recombinant type IV collagen in the preparation of cosmetics for repairing basal barrier and soothing redness; wherein, The recombinant type IV collagen is used as the cosmetic ingredient for repairing the base barrier and soothing and fading redness, and its amino acid sequence is shown in SEQ ID No:

1.

3. The use according to claim 2, wherein The cosmetics are selected from lotions, essences, creams or facial masks.

4. A recombinant collagen composition having the effects of repairing the basal barrier and soothing redness, comprising recombinant type IV collagen and repairing nonapeptide as active ingredients; wherein, In the recombinant collagen composition, the weight ratio of the recombinant type IV collagen to the repair nonapeptide is 500:1 to 10:1; The amino acid sequence of the recombinant type IV collagen is shown in SEQ ID No: 1; The amino acid sequence of the repair nonapeptide is shown in SEQ ID No:

2.

5. The recombinant collagen composition according to claim 4, wherein The weight ratio of the recombinant type IV collagen to the repair nonapeptide is 100:1 to 50:

1.

6. A cosmetic having the effects of repairing the basal barrier and soothing and fading redness, comprising the recombinant collagen composition according to claim 4.

7. The cosmetic according to claim 6, wherein The cosmetic is prepared in a form suitable for application to the skin.

8. The cosmetic according to claim 7, wherein The cosmetics are selected from lotions, essences, creams or facial masks.

9. Use of the recombinant collagen composition according to claim 4 in preparing cosmetics for repairing the basal barrier and soothing redness; wherein, The recombinant type IV collagen and the repairing nonapeptide serve as the cosmetic's functional ingredients for repairing the base barrier and soothing and fading redness.

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