Mesenchymal stem cell secreted peptide and application thereof

Abstract

The application relates to skin care products, in particular to a mesenchymal stem cell secreted peptide and related uses thereof, the secreted peptide obtained by the application has the effects of relieving aging, resisting oxidation, resisting inflammation, and effectively relieving skin damage caused by ultraviolet irradiation.

Description

Technical Field

[0001] This application relates to the field of skin repair and improvement, specifically to a secreted peptide derived from mesenchymal stem cells and its related uses. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell derived from the mesoderm in early development and are among the most studied and important adult stem cells. MSCs are widely distributed in various tissues throughout the body and can be cultured and expanded in vitro. Under specific conditions, they can differentiate into various cell types, including nerve cells, osteoblasts, chondrocytes, muscle cells, and adipocytes. Even after continuous passage and cryopreservation, they retain their multi-lineage differentiation potential. Compared to MSCs from other sources, MSCs derived from the umbilical cord have advantages such as convenient collection, lack of ethical controversy, low immunogenicity, rapid self-renewal, stable doubling rate, and strong proliferative capacity. Therefore, umbilical cord MSCs are suitable for clinical research and application and are the first choice for cell therapy and regenerative medicine.

[0003] according to www.ClinicalTrials.gov Data from the website shows that as of October 2018, 705 MSC-based clinical trials had been registered globally, with over 80% in Phase I and II trials and approximately 15% in Phase III. Current experimental results indicate that MSCs are safe and effective in treating various diseases, including graft-versus-host disease, spinal cord injury, autoimmune diseases, cardiovascular diseases, bone and cartilage repair, Crohn's disease, diabetes and its complications. Currently, 11 mesenchymal stem cell drugs have been approved globally for the clinical treatment of these diseases; however, the mechanisms by which MSCs exert their effects in disease treatment remain unclear and require further in-depth research and exploration.

[0004] Extensive evidence suggests that mesenchymal stem cells (MSCs) exert therapeutic effects through paracrine or autocrine processes, with efficacy related to angiogenesis, immune regulation, and apoptosis. The MSC secretome, the sum of bioactive molecules secreted by MSCs, includes various active components such as cytokines, chemokines, and growth factors, which regulate many physiological processes, including tissue repair, regeneration, and functional recovery. In the field of wound treatment, formulations based on the MSC secretome are highly likely to become alternative therapies to MSC treatment, showing broad development prospects (Wang Qiang et al., Mesenchymal Stem Cell Secretome: An Alternative Therapy for Wound Healing, China Biotechnology Journal 2017, Vol.37 Issue(4):104-109). However, current research on peptides secreted by MSCs is still in its early stages, and current research results are insufficient to meet the diverse needs of consumers. Furthermore, before the MSC secretome can be formally used clinically, more in vivo and in vitro experiments are needed to obtain bioactive peptides with better commercial prospects. Summary of the Invention

[0005] Addressing the shortcomings of existing technologies, the inventors of this application collected cell culture medium, ultrafiltered it at 30kD, and then sent it for mass spectrometry analysis. Peptides were obtained from the mass spectrometry data and screened. A series of peptides were screened from MSC secretions and their functions were verified through cell biology experiments. Bioactive peptides with significant anti-aging, antioxidant, and anti-inflammatory effects were obtained.

[0006] The purpose of this invention is to provide a bioactive polypeptide, its preparation method, and its application.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a bioactive polypeptide LQ-17, the amino acid sequence of which is LSDQVPDTESET. X ILLQ,X is selected from K or R.

[0009] In a second aspect, the present invention provides a method for preparing the bioactive polypeptide LQ-17, which can be artificially synthesized by genetic engineering, directly obtained from cells through isolation and purification, or even more directly prepared by chemical synthesis.

[0010] In a third aspect, the present invention provides the application of the bioactive polypeptide LQ-17 in the preparation of food, health products, pharmaceuticals or cosmetics with anti-inflammatory functions.

[0011] In a fourth aspect, the present invention provides the application of the bioactive polypeptide LQ-17 in the preparation of food, health products or medicines with anti-aging functions.

[0012] In a fifth aspect, the present invention provides the use of the bioactive polypeptide LQ-17 in the preparation of food, health products or pharmaceuticals with antioxidant functions.

[0013] In a sixth aspect, the present invention provides a product comprising the bioactive polypeptide LQ-17 or a derivative thereof; wherein the bioactive polypeptide LQ-17 derivative is a polypeptide derivative obtained by modifying the amino acid side chain groups, amino terminus, or carboxyl terminus of the bioactive polypeptide LQ-17 with hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification, or glycosylation, without affecting its biological activity. Attached Figure Description

[0014] Figure 1 CCK-8 / MTT assay for relative cell proliferation rate

[0015] Figure 2 Relative cell proliferation rate assay after dilution of LQ-17 and DR-13

[0016] Figure 3 Experiment on the inhibition rate of LQ-17ROS

[0017] Figure 4 Experiment on the effect of LQ-17 in reducing inflammatory response

[0018] Figure 5 Experiment on the effects of LQ-17 on normal cellular metabolic activities

[0019] Figure 6 Experiment on the alleviating effect of LQ-17 on UVA damage in cells

[0020] Figure 7 Experimental study on the effect of LQ-17 in reducing collagen damage in cells caused by UVA irradiation.

[0021] Figure 8 Experiment on the effect of LQ-17 in reducing collagen loss caused by ultraviolet radiation Detailed Implementation

[0022] Screening principle:

[0023] The polypeptides involved in this application were obtained using the following procedure:

[0024] Using MSC secretome as experimental material, peptides with molecular weight less than 30 kDa were screened by LC-MS, and 656 bioactive peptides were obtained by ribosome mapping technology. Ten more bioactive peptides were then identified through further screening using a machine learning and biophysics-based protein-peptide interaction prediction (HSM PPI prediction) method, as detailed below:

[0025] Name sequences DE-16 DGQVINETSQHHDDLE EY-13 EALELRDNDKTRY SK-13 SNPVDILTYVAWK NN-16 NTGGSRYPGQGSPGGN AT-17 APEFAALGESGSSSSKT LQ-17 LSDQVPDTESETKILLQ DR-13 DANLEAGNVKETR DG-13 DEAESLEDLGFKG VR-16 VQELQKHQETAEKTKR TT-8 TDLTKVHT

[0026] The following functional verification experiments were conducted on the peptides selected above.

[0027] Experimental steps:

[0028] Cell viability assay

[0029] HaCaT cells were revived and passaged. After microscopic examination showed that the cells were in good condition, the cells were collected and the cell concentration was adjusted to 0.5-1.0 x 10⁻⁶. 5 Cells were seeded at a rate of 100 μL / well in 96-well plates. After incubation for 24 h (37 °C, 5% CO2), a certain concentration of the test substance was added and incubated for another 24 h. The supernatant was removed, and the CCK8 value of the cells was measured using a CCK8 assay kit.

[0030] Antioxidant capacity test

[0031] HaCaT cells were revived and passaged. After microscopic examination showed that the cells were in good condition, the cells were collected. Cells were seeded in 96-well plates for 48 hours, with the cell count controlled at 0.8-1.0 x 10⁻⁶ cells / well. 4 Cells / well. Remove the original culture medium, wash 1-2 times with PBS, add culture medium containing 10 μM H2O2 for 2 h induction, remove the culture medium, add fresh culture medium containing a certain concentration of the test substance, and culture for 24 h. Measure the intracellular ROS level using a ROS detection kit.

[0032] Anti-inflammatory capacity test

[0033] RAW264.7 cells were resuscitated and passaged. After microscopic examination showed that the cells were in good condition, the cells were collected and the cell concentration was adjusted to 1.0-1.5 x 10^6 cells / year. 5 Cells were seeded at a rate of 2 mL / well in 12-well plates. Cells were incubated for 24 hours (37°C, 5% CO2). When the cells reached approximately 80% confluence, the culture medium was discarded, and DMEM high-glucose medium containing LPS was added, along with a specific concentration of the test substance. The plates were incubated for another 24 hours. The supernatant was collected from each well, centrifuged at 12000g for 1 min at 4°C, and the intracellular NO concentration was measured using a NO detection kit.

[0034] Intracellular ATP content detection

[0035] HaCaT cells were resuscitated and passaged. After microscopic examination showed good cell condition, cells were collected and added to 6-well plates at a density of 6*10^5 cells / well. Cells were incubated for 24 hours (37℃, 5% CO2). When the cells reached approximately 80% confluence, the culture medium was discarded, and culture medium containing a certain concentration of the test substance was added. The cells were incubated for another 24 hours. The supernatant was discarded from each well, and 200 μL of lysis buffer was added. The cell lysis products were collected, centrifuged at 12000g for 1 min at 4℃, and the supernatant was used to determine the ATP concentration using an ATP assay kit.

[0036] UV resistance test

[0037] Human foreskin fibroblasts (HFF cells) were resuscitated and passaged. After microscopic examination showed that the cells were in good condition, the cells were collected and cultured at a density of 2.0-2.5 x 10⁻⁶ cells / mL. 4 Cells were added to 96-well plates at a density of [number] cells / well. The plates were incubated for 24 hours (37℃, 5% CO2), the culture medium was discarded, and a culture medium containing a certain concentration of the test substance was added. Induction was performed using a specific dose of UVA, ensuring the total irradiation was consistent and between 5-15 J / cm². 2 Discard the supernatant, add culture medium containing a certain amount of the test substance, and incubate for 24 hours. Then, use the corresponding kit to determine the ROS content, CCK8, hydroxyproline content, and other indicators.

[0038] Experimental results:

[0039] 1. Human keratinocyte HaCaT cells were treated without damage. A peptide at a concentration of 100 ppm was added, and changes in CCK8 levels were detected to assess cell viability. NC (no cell count) served as the negative control, containing only cells and no sample. Statistical analysis (mean ± SD, n = 6) was performed using an unpaired t-test to compare with NC. Cell viability results (e.g.) Figure 1 The results showed that LQ-17 and DR-13 could enhance cell viability. Gradient experiments were conducted with decreasing concentrations of LQ-17 and DR-13 (100, 50, 10 ppm). CCK8 assays were repeated. NC (cell-only) was used as the negative control, containing only cells and no additional sample. Statistical analysis (mean ± SD, n = 6) was performed using the unpaired t-test to compare with NC. Experimental results (see [link to results]). Figure 2 The results showed that at all concentrations, LQ-17 had a better anti-aging effect than DR-13, and even at an extremely low concentration level of 10 ppm, it could still increase cell activity by about 10%.

[0040] 2. Hydrogen peroxide was used to induce an increase in the oxidation level of HaCaT cells. A peptide concentration of 10 ppm was added. VC (vitamin C) was used as the positive control group (100 μM); CK (cells only, no induction treatment) was used as the blank control group; NC (cells induced with hydrogen peroxide) was used as the negative control group. Statistical analysis (mean ± SD, n = 6) was performed using the unpaired t-test for comparison with NC. Experimental results showed (e.g.) Figure 3 LQ-17 can significantly reduce intracellular ROS levels, which is superior to other peptides, and its efficacy is comparable to that of vitamin C.

[0041] 3. Intracellular inflammation in RAW264.7 cells was induced using LPS at a final concentration of 1 μg / mL. The peptide concentration was 10 ppm. PC (positive control) was supplemented with LPS + dexamethasone (100 μg / mL); NC (negative control) was supplemented with LPS; CK (blank control) was supplemented with cells only. Statistical analysis (mean ± SD, n = 2) was performed using the unpaired t-test to compare with NC. The results showed that LQ-17 significantly reduced NO concentration (approximately 20% decrease compared to the negative control) and significantly alleviated the inflammatory response (see [link to relevant documentation]). Figure 4 ).

[0042] 4. Add 10 ppm of LQ-17 to HaCaT cells. EGF, positive control, added at a concentration of 1 nM; CK, blank control, added only cells. Statistical analysis (mean ± SD, n = 2) was performed using the Unpaired t-test for comparison with NC. Experimental results (see [link to results]). Figure 5 LQ-17 has no effect on ATP production, meaning that LQ-17 does not affect the cell's main energy metabolism activities.

[0043] 5. LQ-17 peptide was added to UVA-induced human foreskin fibroblasts (HFF and HaCaT cells) at a concentration of 10 ppm. Vitamin C (100 μM) was used as the positive control; TGF-β (100 ng / mL) as the positive control; CK (cells only, without induction treatment) was used as the blank control; and NC (cells treated with UVA induction) was used as the negative control. Statistical analysis (mean ± SD, n = 3) was performed using the unpaired t-test compared to NC. The experimental results showed that:

[0044] 1. Compared with the negative control, LQ-17 significantly improved cell survival rate after UV irradiation. See details below. Figure 6 The left image shows HFF cells; the right image shows HaCaT cells.

[0045] 2. Compared with the negative control, LQ-17 significantly reduced the ROS concentration increased by UV irradiation. See details below. Figure 7 The left image shows HFF cells; the right image shows HaCaT cells.

[0046] 10 ppm of LQ-17 peptide was added to UVA-induced HFF cells. TGF-β was used as the positive control (100 ng / mL); CK was used as the blank control (cells without induction treatment); and NC was used as the negative control (cells treated with UVA). Statistical analysis was performed using the unpaired t-test (mean ± SD, n = 6) to compare with NC. Experimental results showed (see [link to results]). Figure 8 LQ-17 peptide can promote the increase of extracellular hydroxyproline content in cells damaged by UVA irradiation, reduce collagen loss caused by ultraviolet irradiation, delay skin aging, and reduce skin damage.

[0047] Based on the above experimental data, LQ-17 peptide exhibits excellent effects in anti-aging, anti-oxidation, reducing inflammation, and reducing skin damage, with no toxic side effects. Furthermore, the LQ-17 peptide used in this series of experiments is LSDQVPDTESETKILLQ, whose human-derived LQ-17 peptide (LSDQVPDTESETRILLQ) showed similar effects in anti-aging, anti-oxidation, reducing inflammation, and reducing skin damage in the aforementioned experiments. SEQUENCE LISTING <110>㽭 <120> <130> HM2025 <160> 10 <170> PatentIn version 3.3 <210> 1 <211> 16 <212> PRT <213> <400> 1 Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp Leu Glu 1 5 10 15 <210> 2 <211> 13 <212> PRT <213> <400> 2 Glu Ala Leu Glu Leu Arg Asp Asn Asp Lys Thr Arg Tyr 1 5 10 <210> 3 <211> 13 <212> PRT <213> <400> 3 Ser Asn Pro Val Asp Ile Leu Thr Tyr Val Ala Trp Lys 1 5 10 <210> 4 <211> 16 <212> PRT <213> <400> 4 Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn 1 5 10 15 <210> 5 <211> 17 <212> PRT <213> <400> 5 Ala Pro Glu Phe Ala Ala Leu Gly Glu Ser Gly Ser Ser Ser Ser Lys 1 5 10 15 Thr <210> 6 <211> 17 <212> PRT <213> <400> 6 Leu Ser Asp Gln Val Pro Asp Thr Glu Ser Glu Thr Lys Ile Leu Leu 1 5 10 15 Gln <210> 7 <211> 13 <212> PRT <213> <400> 7 Asp Ala Asn Leu Glu Ala Gly Asn Val Lys Glu Thr Arg 1 5 10 <210> 8 <211> 13 <212> PRT <213> <400> 8 Asp Glu Ala Glu Ser Leu Glu Asp Leu Gly Phe Lys Gly 1 5 10 <210> 9 <211> 16 <212> PRT <213> <400> 9 Val Gln Glu Leu Gln Lys His Gln Glu Thr Ala Glu Lys Thr Lys Arg 1 5 10 15 <210> 10 <211> 8 <212> PRT <213> <400> 10 Thr Asp Leu Thr Lys Val His Thr 1 5

Claims

1. A mesenchymal stem cell secreted peptide having the sequence: LSDQVPDTESET X ILLQ, X is selected from K or R.

2. Use of the secretion peptide of claim 1 in the preparation of a cosmetic or pharmaceutical for anti-aging function.

3. Use of the secretion peptide of claim 1 in the preparation of a cosmetic or pharmaceutical for anti-UV damage function.

4. Use of the secretion peptide of claim 1 in the preparation of a cosmetic or pharmaceutical for anti-inflammatory function.

Images