Recombinant IV type collagen composition for repairing basement membrane and relieving and fading red

Abstract

The invention provides a recombinant IV type collagen composition for repairing a basement membrane and relieving and fading red. The inventor finds that the recombinant IV-type collagen disclosed by the patent specification inhibits VE-cadherin protein phosphorylation through a mediated PKC alpha / PYK2 / Src signal channel, protects tight connection between endothelial cells, prevents blood component leakage and reduces vascular permeability, and the recombinant IV-type collagen has the advantages that the recombinant IV-type collagen can inhibit the VE-cadherin protein phosphorylation through the mediated PKC alpha / PYK2 / Src signal channel; the dissociation of the VE-cadherin compound caused by oxidative stress can be improved, and the steady state of an endothelial barrier is maintained; therefore, the composition has good effects of repairing a basement membrane barrier, relieving and fading red. The inventor also finds that the repairing nonapeptide (GAAGLPGPK) has the effects of repairing the basal membrane barrier, relieving and fading red for the first time, and the combination of the recombinant IV type collagen and the nonapeptide in a certain proportion shows synergistic interaction in the aspects of repairing the basal membrane barrier, relieving and fading red.

Description

Technical Field

[0001] The present invention relates to the technical field of functional cosmetics or external medicines, and specifically to recombinant type IV collagen and repair functional peptides with the functions of repairing basement membrane barriers and soothing and fading redness, and their use in preparing cosmetics or external medicines with the functions of repairing basement membrane barriers and soothing and fading redness. Background Art

[0002] As an important structural protein, collagen 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 pathogens and large differences between batches.

[0003] In the field of skin health and repair, the basement membrane barrier plays a vital role. It not only maintains the structural integrity of the skin, but also participates in the regulation of many physiological functions. However, in daily life, the skin is frequently attacked by harmful external factors, such as ultraviolet rays (UVA and UVB), which can damage the basement membrane barrier and significantly reduce the content of important basement membrane proteins such as laminin, thereby destroying the normal physiological functions of the skin and causing a series of skin problems, such as sagging skin, wrinkles, and accelerated water loss.

[0004] At the same time, the endothelial barrier also faces many challenges in the body. Reactive oxygen species such as hydrogen peroxide (H2O2) can be produced by the body's metabolic process or external stimuli. It can destroy the integrity of endothelial cell adhesion connections, specifically by promoting the disintegration of the VE-cadherin complex and causing damage to the endothelial barrier function. This damage interferes with the tight junctions between cells, leading to abnormal permeation of substances, affecting the normal homeostasis of the tissue's internal environment, and is closely related to the occurrence and development of a variety of inflammatory and vascular-related diseases.

[0005] In addition, the number of people with sensitive skin is increasing, and their skin is prone to discomfort symptoms such as redness and stinging, which not only affects appearance but also reduces the quality of life. Although existing technologies have some countermeasures for these skin problems, there is still a lack of efficient and targeted solutions in terms of repairing the basement membrane barrier, protecting the endothelial barrier, and soothing sensitive skin, making it difficult to meet people's urgent needs for skin health maintenance and repair. Summary of the invention

[0006] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a recombinant type IV collagen with good effects of repairing the basal barrier and soothing and fading redness, a small peptide that synergizes with the recombinant type IV collagen, and their use in the preparation of cosmetics or external medicines for repairing the basal barrier and soothing and fading redness.

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

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

[0009] 1. A use of recombinant type IV collagen in the preparation of cosmetics or external medicines for repairing the basal barrier and soothing redness; wherein the recombinant type IV collagen is used as an effective ingredient for repairing the basal barrier and soothing redness of the cosmetics or external medicines, and its amino acid sequence is shown in SEQ ID No: 1.

[0010] SEQ ID No:1

[0011] GPPGPPGIVIGTGPLGEKGERGYPGTPGPRGEPGPKGFPGLPGQPGPPGLPVPGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLPGPPGSPGPPGQPGYTNGIVECQPGPPGDQGPPGIPGQPGFIGEIGEKGQKGE SCLICDIDGYRGPPGPQGPPGEIGFPGQPGAKGDRGLPGRDGVAGVPGPQGTPGLIGQPGAKGEPGEFYFDLRLKGDKGDPGFPGQPGMPGRAGSPGRDGHPGLPGPKGSPGSVGLKGERGPPGGVGFPGSRGDTGPPGPP

[0012] 2. The use according to item 1, wherein the cosmetic or external medicine 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 facial mask.

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

[0015] SEQ ID No:2

[0016] GAAGLPGPK

[0017] 5. A recombinant collagen composition having the effects of repairing the basal barrier and soothing and fading 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 repairing nonapeptide is 500:1 to 10:1;

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

[0019] The amino acid sequence of the repair nonapeptide is 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 repair nonapeptide is 50:1 to 100:1.

[0021] 7. A cosmetic or external medicine having the effects of repairing the basal barrier and soothing and fading redness, comprising 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 facial mask.

[0024] 10. Use of the recombinant collagen composition according to item 4 in the preparation of cosmetics or external medicines for repairing the basal barrier and soothing redness; wherein the recombinant type IV collagen and the repairing nonapeptide serve as the cosmetics or external medicines for repairing the basal barrier and soothing redness.

[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] Effects of the Invention

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

[0029] The results of the mechanism research experiments showed 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 junction protein complex between endothelial cells, and protect the endothelial barrier function. The results of the human efficacy evaluation showed that the recombinant type IV collagen has the effect of significantly improving skin redness. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0033] Figure 4 The figure shows the changes of skin hemoglobin EI value at different time points.

[0034] *Indicates significant difference compared with Do group, ***P<0.001

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

[0036] *Indicates significant difference compared with Do group, ***P<0.001

[0037] Figure 6 This is a graph showing the changes in the red zone area at different time points.

[0038] *Indicates significant difference compared with Do group, **P<0.01, ***P<0.001 DETAILED DESCRIPTION

[0039] Example 1 Preparation of recombinant type IV collagen and repair nonapeptide

[0040] With reference to Example 1 and Example 2 of the specification of Chinese patent application publication CN117903287A, the recombinant type IV collagen was prepared and purified.

[0041] The repair nonapeptide was commissioned to Gill Biochemical (Shanghai) Co., Ltd. for synthesis. The peptide sequence was GAAGLPGPK. Its amino acid sequence and purity were determined by mass spectrometry and high performance liquid chromatography.

[0042] Example 2: Effect of recombinant type IV collagen, repairing nonapeptide 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 at 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), add 2 mL to each well, and culture in a 37°C, 5% CO2 incubator. After 24 hours of culture, irradiation and sample intervention began. 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 (30J / cm 2 ) and UVB (75mJ / cm 2 ), after irradiation, the model group was replaced with fresh culture medium, and the positive control group and sample group were replaced with fresh culture medium containing samples for intervention treatment, and the amount of liquid added to each well was 2mL. The normal group only replaced the culture medium, without irradiation and sample intervention.

[0046] Immunofluorescence detection of laminin: After PBS washing, fixation with 4% paraformaldehyde was performed for 15 minutes, and then immunofluorescence detection was performed. Observation was performed under a microscope, and pictures 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 combined drug index CI = (A × B) / AB for each sample group. 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. Among them,

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

[0049] T = laminin amount in each sample group minus laminin amount in the model control group, representing the actual level of laminin recovery 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 represent the (1+T / C) values ​​when recombinant type IV collagen or repair nonapeptide was administered alone, respectively;

[0052] AB represents the (1+T / C) value when Compositions 1 to 5 were 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 (n = 3)

[0056]

[0057] Compared with the normal group, the laminin content in the model group decreased significantly, indicating that the stimulation conditions of this experiment were effective and the modeling was successful. Compared with the model group, the laminin content of each sample group increased significantly, indicating that each sample has a significant effect in repairing the basement membrane barrier. The combined medication index of the above-mentioned compositions 2, 3, 4 and 5 is less than 1, indicating that the recombinant type IV collagen: repair nine peptides have a synergistic effect when the weight ratio is 500 to 10:1. Moreover, the synergistic index of compositions 3 and 4 is less than 0.9, indicating that the synergistic effect of recombinant type IV collagen: repair nine peptides is very significant when the ratio is 100 to 50:1. Among them, the synergistic effect of composition 3 is the most significant.

[0058] This shows that recombinant type IV collagen, repairing nonapeptide and their combination can promote the expression of important proteins in the basement membrane after damage, and have the effect of repairing the basement membrane barrier and soothing redness.

[0059] Example 3: Study on the maintenance of homeostasis of proteins related to endothelial cell adhesion junctions 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, cultured overnight, and the cell confluence reached 60% to 70%. Different concentrations of recombinant type IV collagen (0.025, 0.05, and 0.1 mg / mL) were added to treat the cells, and a normal group (C) and a model group (M) were set up. After 24 hours, HUVEC cells were lysed to obtain protein samples according to the manufacturer's instructions of the total protein extraction kit (BC3711, Solarbio, China). Equal amounts of protein samples were taken and separated on a 7.5% SDS-PAGE gel, and transferred to a 0.45 μM PVDF membrane using different currents according to the size of the protein molecules. The membrane was further blocked with 5% skim milk. The membrane was then incubated with primary antibodies for 16 h at 4°C, including β-actin, PKCα, phosphorylated PKCα, Src, phosphorylated Src, PYK2, phosphorylated PYK2, VE-cadherin, and phosphorylated VE-cadherin. Horseradish peroxidase-conjugated affinity pure goat anti-rabbit IgG (H+L) (SA00001-2, ) to detect the antigen and antibody complex. Finally, an enhanced chemiluminescence kit was used for color development, and the immunoreactive bands were scanned and quantitatively analyzed by ImageJ software. This experiment can detect the expression levels of various proteins and their phosphorylation states under different treatment conditions, which is helpful for analyzing protein expression and signal pathway activation.

[0062] Co-immunoprecipitation analysis:

[0063] First, HUVEC cells were lysed, and the supernatant was collected by adding appropriate amounts of IP cell lysate and protease inhibitors. A small amount of 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 common IgG of the same species as the precipitation antibody used in the IP experiment and 20 μL of protein A / G magnetic beads (mixed thoroughly 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 incubated at 4°C for 1 hour with shaking. The remaining supernatant was removed, placed in a new tube, and incubated with VE-cadherin antibody at 4°C for 2 hours with continuous stirring. Then, protein A / G magnetic beads were added to the supernatant / antibody mixture and incubated overnight at 4°C with continuous stirring. The magnetic beads were washed three times with ice-cold IP lysis buffer. The bound protein was eluted with 1× protein sample buffer and subjected to SDS-PAGE electrophoresis. Finally, Western Blot experiments were performed 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 β-catenin.

[0064] Experimental conclusion:

[0065] Table 2 Quantitative analysis of immunoblotting

[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 effect of recombinant type IV collagen on PKCα / Src / PYK2 / VE-cadhrein and its phosphorylated proteins. The protein electrophoresis diagram is shown in Figure 2 As 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. STDEV and T test were calculated. 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 h, VE-cadherin was immunoprecipitated, and the presence of p120-catenin and β-catenin in the immunoprecipitates was assessed ( 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 reduced. The cells were treated with recombinant type IV collagen for 24 hours before modeling, and a dose-dependent increase in p120-catenin and β-catenin was detected. Recombinant type IV collagen inhibited the H2O2-induced dissociation of β-catenin and p120-catenin from the VE-cadherin complex. These results indicate that H2O2 destroys the integrity of endothelial cell adhesion junctions, leading to the disintegration of the VE-cadherin complex, while recombinant type IV collagen protects the VE-cadherin complex, maintains the interaction between adhesion junction-related proteins, strengthens the connection between endothelial cells, and thus protects the integrity of the endothelial barrier.

[0070] In general, the results of immunoprecipitation and Western Blot confirmed each other, and elaborated in detail from the molecular level the destructive mechanism of H2O2 on endothelial barrier-related proteins and complexes, as well as the protective mechanism of recombinant type IV collagen, providing a solid basis for a deep understanding of the cellular biological basis behind the problem of skin soothing and fading redness, and for the development of targeted treatment strategies.

[0071] Example 4: Evaluation of the efficacy of recombinant type IV collagen and composition 3 in soothing and fading redness in humans

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

[0073] Experimental conclusion:

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

[0075] In addition, the skin color (red-green) values ​​(a* values) of the 30 subjects were significantly different after 14 and 28 days of using recombinant type IV collagen or composition 3 (sample group), while there was no significant difference in a* values ​​after 14 and 28 days of using a matrix without recombinant type IV collagen or composition 3 (control group). After 14 days, the a* values ​​of 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 between the groups; after 28 days, the a* values ​​of 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 between the groups ( Figure 5 ). This again shows that both recombinant type IV collagen and composition 3 have soothing effects after 14 days and 28 days of use, and 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 area of ​​redness and swelling before use, the 30 subjects showed significant differences in the area of ​​redness and swelling after 14 and 28 days of using recombinant type IV collagen or composition 3 (sample group), while there was no significant difference in the area of ​​redness and swelling after 14 and 28 days of using the matrix without recombinant type IV collagen or composition 3 (control group). After 14 days of using recombinant type IV collagen, the area of ​​redness and swelling in the sample group decreased by 10.55%, the area of ​​redness and swelling in the composition 3 group decreased by 14.98%, and the area of ​​redness and swelling in the control group decreased by 0.11%, with significant differences between the two groups. After 28 days of use, the area of ​​redness and swelling in the recombinant type IV collagen group decreased by 9.75%, the area of ​​redness and swelling in the composition 3 group decreased by 16.04%, and the area of ​​redness and swelling in 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 at both 14 and 28 days of use, and the soothing effect of the sample group is superior to 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 and 28 days of use.

Claims

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

1.

2. Use of recombinant type IV collagen in the preparation of cosmetics or external medicines for repairing basal barrier and relieving redness; wherein: The recombinant type IV collagen is used as a component of the cosmetic or external medicine 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 1, wherein The cosmetics are selected from lotions, essences, creams or facial masks.

4. A polypeptide having the activity of repairing basal barrier and soothing redness fading, the amino acid sequence of which is shown in SEQ ID No:

2.

5. A recombinant collagen composition having the effects of repairing the basal barrier and soothing and fading 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.

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

1.

7. A cosmetic or external medicine having the effects of repairing the basal barrier and soothing and fading redness, comprising the recombinant collagen composition described in item 5.

8. The cosmetic or external medicine according to claim 7, wherein: The cosmetic or external medicine is prepared in a form suitable for application to the skin.

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

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

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