Composition capable of resisting inflammation and promoting skin repair as well as preparation method and application of composition

Abstract

The invention discloses a composition capable of resisting inflammation and promoting skin repair as well as a preparation method and application of the composition. The composition comprises, in addition to platelet rich plasma (PRP), beta-nicotinamide mononucleotide (NMN) and sialic acid (NANA). The composition of the platelet-rich plasma, the nicotinamide mononucleotide and the sialic acid, provided by the invention, can obviously accelerate the wound healing speed, the wound healing speed is faster than that of the single use of the platelet-rich plasma, the nicotinamide mononucleotide or the sialic acid, and the scar color after wound scar repair is light.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a composition capable of resisting inflammation and promoting skin repair, and a preparation method and application thereof. In addition to platelet-rich plasma, the composition also contains β-nicotinamide mononucleotide and sialic acid, and can be applied to skin scalds, burns, atopic dermatitis, diabetic foot and skin anti-aging. Background Art

[0002] There is a certain correlation between blood, β-nicotinamide mononucleotide (NMN) and telomeres. Cells in the blood contain telomeres, and NMN can affect the length and stability of telomeres. Studies have shown that the length and stability of telomeres in cells in the blood are related to the risk of various diseases. For example, proteins in plasma: FGF, IGF-1, TNF-α, etc. have also been found to be related to the length and stability of telomeres. Studies have found that NMN can increase the content of NAD+ in mouse skin cells, enhance the energy metabolism and antioxidant capacity of cells, and thus promote wound healing. In addition, NMN can also promote wound healing through mechanisms such as repairing DNA damage, stimulating cell proliferation and regulating immune responses, but related clinical applications are relatively rare.

[0003] Platelet-rich plasma (PRP) refers to platelet concentrate extracted from autologous blood by centrifugation. It can promote the repair of bones and soft tissues and has been used in orthopedics, oral and maxillofacial surgery, cardiothoracic surgery, neurosurgery, obstetrics and gynecology, ophthalmology, otolaryngology, general surgery, and plastic surgery.

[0004] Sialic acid, also known as bird's nest acid, is the main component of bird's nest. It can inhibit the activity of tyrosine hydroxylase and dopa hydroxylase, prevent the formation of melanin, and lighten facial spots. Due to the presence of carboxyl groups in sialic acid molecules, they carry negative charges, which is beneficial for cells to participate in water-salt balance, material transport and other processes, and is very important in ensuring the normal function of cells.

[0005] However, the prior art is still lacking a composition that can resist inflammation and promote skin repair, which can make wounds heal faster and make scars lighter in color after wound scar repair, when used for skin repair, including skin burns, atopic dermatitis, diabetic foot and skin anti-aging. Summary of the invention

[0006] The technical problem to be solved by the present invention is that the prior art lacks an anti-inflammatory and skin repair-promoting composition that can make wounds heal faster and make scars lighter in color after wound scar repair when used for skin repair, including skin burns, atopic dermatitis, diabetic foot and skin anti-aging. Thus, a composition that can resist inflammation and promote skin repair, and a preparation method and application thereof are provided.

[0007] The present invention refers to the composition of sialic acid, NMN and platelet-rich plasma as PRP plus, which promotes wound healing of mouse skin by inhibiting inflammatory factors and promoting cell migration of skin keratinocytes HaCaT. The composition has anti-inflammatory, antioxidant and wound healing effects. Compared with PRP, PRP plus has better anti-inflammatory, antioxidant and mouse wound healing effects than PRP. PRP plus can be used for skin repair, including skin burns, atopic dermatitis, diabetic foot and skin anti-aging, and its function has been verified by actual cases.

[0008] Based on the above technical problems, one of the technical solutions provided by the present invention is: a composition capable of resisting inflammation and promoting skin repair, wherein the composition contains, in addition to platelet-rich plasma, β-nicotinamide mononucleotide and sialic acid.

[0009] In a specific embodiment of the present invention, the volume-to-mass ratio of platelet-rich plasma, sialic acid and β-nicotinamide mononucleotide in the composition is (100-10000):1:1 (V / W / W).

[0010] In a specific embodiment of the present invention, the volume-to-mass ratio of platelet-rich plasma, sialic acid and β-nicotinamide mononucleotide in the composition is (1000-5000):1:1 (V / W / W).

[0011] In a specific embodiment of the present invention, the volume-to-mass ratio of platelet-rich plasma, sialic acid and β-nicotinamide mononucleotide in the composition is (2500-3500):1:1 (V / W / W).

[0012] In a specific embodiment of the present invention, the volume-to-mass ratio of platelet-rich plasma, sialic acid and β-nicotinamide mononucleotide in the composition is 3000:1:1 (V / W / W).

[0013] In the present invention, the platelet-rich plasma may be derived from human or non-human animals.

[0014] In a specific embodiment of the present invention, the platelet-rich plasma is from humans, deer, cattle, horses, sheep, pigs, chickens, rabbits or fish.

[0015] Based on the above technical problems, the second technical solution provided by the present invention is: a method for preparing the composition as described in the first technical solution, the preparation method comprising uniformly mixing platelet-rich plasma, β-nicotinamide mononucleotide and sialic acid to prepare the composition.

[0016] In a specific embodiment of the present invention, the uniform mixing is performed by ultrasonication at 90 to 110 W for 8 to 12 minutes.

[0017] In a specific embodiment of the present invention, the uniform mixing is 100W ultrasound for 10 minutes.

[0018] In a specific embodiment of the present invention, the platelet-rich plasma is obtained by centrifugation from fresh blood isolated from the body.

[0019] In a specific embodiment of the present invention, the centrifugal separation is differential centrifugation, which includes centrifuging the fresh blood at 1200-2000 rpm for 4-6 minutes and then at 3000-3800 rpm for 18-22 minutes.

[0020] In a specific embodiment of the present invention, the differential centrifugation comprises centrifuging the fresh blood at 1500 rpm for 5 minutes and then at 3500 rpm for 20 minutes.

[0021] In a specific embodiment of the present invention, the preparation method further comprises freeze-drying the uniformly mixed mixture to obtain the composition.

[0022] In a specific embodiment of the present invention, the freeze-drying temperature is -80°C.

[0023] Based on the above technical problems, the third technical solution provided by the present invention is: use of the composition described in the first technical solution in the preparation of skin repair drugs or cosmetics.

[0024] In a specific embodiment of the present invention, the skin repair comprises one or more selected from the group consisting of skin regeneration, skin anti-aging and assisting skin wound healing.

[0025] In a specific embodiment of the present invention, the skin trauma includes one or more selected from skin scald, skin burn, atopic dermatitis and diabetic foot; the skin anti-aging includes skin anti-oxidation.

[0026] In a specific embodiment of the present invention, the concentration of the composition in the medicine or cosmetic is 0.1-5.0% (V / V), preferably 0.5-1.5% (V / V).

[0027] In a specific embodiment of the present invention, the concentration of the composition in the medicine or cosmetic is 1.0% (V / V).

[0028] The concentration can be adjusted using any suitable solvent in the art, for example, physiological saline can be used to adjust the composition to the concentration.

[0029] Based on the above technical problems, the fourth technical solution provided by the present invention is: the composition as described in the first technical solution is used for skin repair.

[0030] In a specific embodiment of the present invention, the skin repair comprises one or more selected from the group consisting of skin regeneration, skin anti-aging and assisting skin wound healing.

[0031] In a specific embodiment of the present invention, the skin trauma includes one or more selected from skin scald, skin burn, atopic dermatitis and diabetic foot; the skin anti-aging includes skin anti-oxidation.

[0032] In a specific embodiment of the present invention, the composition is administered at a concentration of 0.1-5.0% (V / V), preferably 0.5-1.5% (V / V).

[0033] In a specific embodiment of the present invention, the composition is administered at a concentration of 1.0% (V / V).

[0034] Any suitable solvent in the art may be used to adjust the concentration, for example, physiological saline may be used to adjust the composition to the administration concentration.

[0035] Based on the above technical problems, the fifth technical solution provided by the present invention is: a method for skin repair, which comprises administering a therapeutically effective amount of the composition as described in one of the technical solutions to a patient in need.

[0036] In a specific embodiment of the present invention, the skin repair comprises one or more selected from the group consisting of skin regeneration, skin anti-aging and assisting skin wound healing.

[0037] In a specific embodiment of the present invention, the skin trauma includes one or more selected from skin scald, skin burn, atopic dermatitis and diabetic foot; the skin anti-aging includes skin anti-oxidation.

[0038] In a specific embodiment of the present invention, the composition is administered at a concentration of 0.1-5.0% (V / V), preferably 0.5-1.5% (V / V).

[0039] In a specific embodiment of the present invention, the composition is administered at a concentration of 1.0% (V / V).

[0040] Any suitable solvent in the art may be used to adjust the concentration, for example, physiological saline may be used to adjust the composition to the administration concentration.

[0041] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0042] The human blood used in this study was obtained from the Hong Kong Red Cross, and the study was approved by the Ethics Committee of the Hong Kong University of Science and Technology.

[0043] The reagents and raw materials used in the present invention are commercially available.

[0044] Compared with the traditional technical solution, the present invention has the following advantages:

[0045] The present invention adds nicotinamide mononucleotide (NMN) on the basis of traditional PRP technology, which can not only stabilize the telomeres of blood cells, but also directly contact the injured skin cells, and convert them into NAD+ more directly and efficiently, thereby enhancing the energy metabolism and antioxidant capacity of cells, stimulating cell proliferation and regulating immune response and other mechanisms to promote wound healing; sialic acid molecules carry a negative charge, which is beneficial for cells to participate in physiological processes such as water-salt balance and material transport, and can effectively inhibit the activity of tyrosine hydroxylase and dopa hydroxylase, preventing the formation of melanin, and mainly plays the role of lightening the color of spots and wound scars in the present invention; platelet-rich plasma contains rich cytokines and growth factors, which play a very important role in the process of wound healing.

[0046] The composition of platelet-rich plasma, nicotinamide mononucleotide (NMN) and sialic acid provided by the present invention can significantly accelerate the wound healing speed, which is faster than the wound healing using platelet-rich plasma PRP, nicotinamide mononucleotide (NMN) or sialic acid alone; and the scar color after the wound scar is repaired is lighter. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 This is a test of the toxicity of PRP and PRP plus on HaCaT cells, which shows the effects of different concentrations of PRP plus and PRP on the viability (%) of HaCaT cells.

[0049] Figure 2 Comparison of the HaCaT cell migration assay results of PRP plus, PRP, sialic acid, and NMN.

[0050] Figure 3 Comparison of different ratios of PRP plus composition, PRP, sialic acid and NMN in inhibiting melanin production, compared with the control group: **P<0.01.

[0051] Figure 4 Comparison of the effects of PRP plus, PRP, sialic acid and NMN on the gene expression of collagen COL1A1, compared with the control group: *P<0.05, **P<0.01.

[0052] Figure 5 Comparison of the inhibitory effects of PRP plus, PRP, sialic acid and NMN on IL-6 inflammatory factor, compared with the control group: *P<0.05, **P<0.01.

[0053] Figure 6 Comparison of the inhibitory effects of PRP plus, PRP, sialic acid and NMN on TNF-α inflammatory factors, compared with the control group: ***P<0.001.

[0054] Figure 7 Comparison of the inhibitory effects of PRP plus, PRP, sialic acid and NMN on COX2 inflammatory factors, compared with the control group: ***P<0.001.

[0055] Figure 8 Comparison of the inhibitory effects of PRP plus, PRP, sialic acid and NMN on iNOS, compared with the control group: *P<0.05, **P<0.01, ***P<0.001.

[0056] Fig. 9 Comparison of the antioxidant efficacy of different ratios of PRP plus compositions, PRP, sialic acid, and NMN.

[0057] Fig.10 This is a wound healing experiment using blood from different animals.

[0058] Fig.11 PRP and PRP plus promote wound healing in mice.

[0059] Fig.12 The results of wound healing in children with burns.

[0060] Fig.13 This is a picture showing the effect of improving scars.

[0061] Fig.14 This is a diagram showing the effect of promoting wound healing in diabetic foot.

[0062] Fig.15 This is a diagram showing the effect of promoting wound healing in diabetic foot. DETAILED DESCRIPTION

[0063] For ease of understanding of the present invention, the present invention will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0064] Example 1: Preparation of platelet-rich plasma and its composition

[0065] The specific steps are as follows:

[0066] 1) Fresh blood (human blood used in this study was obtained from the Hong Kong Red Cross) was collected and centrifuged at two different speeds (1200-2000 rpm for 5 minutes and 3000-3800 rpm for 20 minutes) to obtain platelet-rich plasma (PRP).

[0067] 2) NMN (purchased from Jinan World Chemical Co., Ltd.) and sialic acid (purchased from Jiabiyu Biotechnology (Wuhan) Co., Ltd.) were added to platelet-rich plasma in a certain proportion (the volume mass ratio of platelet-rich plasma, sialic acid and β-nicotinamide mononucleotide was 100:1:1 (V / W / W), 1000:1:1 (V / W / W), 3000:1:1 (V / W / W), 5000:1:1 (V / W / W) or 10000:1:1 (V / W / W)), ultrasonically treated (100W) for 10 minutes, mixed evenly, and stored in a -80°C low-temperature refrigerator; freeze-dried for standby use, called PRP plus.

[0068] Example 2: Cytotoxicity test

[0069] In order to evaluate whether the PRP plus of the present invention has cytotoxicity in stratum corneum cells and whether it will stimulate skin cell proliferation, the MTT method was used to detect and analyze the cell survival rate. HaCaT keratinocytes (catalog number T0020001, purchased from AddexBio) were cultured in DMEM medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin. The cultured cells were digested with 0.25% trypsin solution to obtain the adherent cells at the bottom of the culture dish, and a cell suspension was prepared and counted, and then a cell concentration of 1×10 4 The suspension of 1000 cells / mL was inoculated on a 96-well plate and placed in a CO2 incubator (5% CO2, 37°C) for 24 hours. Then different concentrations of PRP and PRP plus were added for treatment. After 24 hours of continuous culture, the medium in the 96-well plate was removed, and 0.5 mg / mL of MTT solution was added. The plate was placed in an incubator for 4 hours. After the culture was completed, DMSO solution was added and shaken on a shaker for 15 minutes. The absorbance at 570 nm was measured to analyze the cell viability.

[0070] The results are as follows Figure 1As shown, both PRP plus and PRP have no cytotoxicity to HaCaT skin keratinocytes. If the concentration exceeds 5% (v / v with culture medium), the cell culture medium becomes gel-like and is not suitable for experiments such as scratch cell migration. The optimal concentration of the experiment is 1% (v / v). In other embodiments, PRP plus and PRP are both used at a concentration of 1% (v / v).

[0071] Example 3: Wound healing experiment

[0072] In this example, skin keratinocytes HaCaT were used to conduct wound healing experiments on the 1% PRP plus composition prepared according to the method of Example 1, 1% PRP, 1 mM NMN and 1 mM sialic acid.

[0073] The specific experimental steps are as follows:

[0074] 1) HaCaT cells were cultured in DMEM medium containing 10% (v / v) FBS (fetal bovine serum) and 1% (w / v) P / S (penicillin / streptomycin). The adherent cells at the bottom of the culture dish were digested with 0.25% (w / v) trypsin solution to prepare a cell suspension, which was counted and diluted to a cell concentration of 1×10 5 The suspension was inoculated into a 12-well plate and placed in a CO2 incubator (5% CO2, 37°C) until the cells were completely densely spread.

[0075] 2) Use a 200 μL pipette tip to draw a cross at the center of the bottom of each well of the 12-well plate to simulate wound formation, and photograph the cell layer coverage under a 50x magnification microscope for record, which is designated as T0.

[0076] 3) Add blank control (no other ingredients except culture medium), positive control (VEGF, vascular endothelial growth factor), PRP, PRP plus and NMN to the 12-well plate. After adding the sample and culturing for 20 hours, take another picture of the cell layer coverage and set it as T20. The results are shown in Figure 2 .

[0077] Figure 2 This is an experimental diagram of the cell layer coverage of keratinocyte HaCaT cells. From left to right, they correspond to blank control, vascular endothelial growth factor VEGF, PRP plus, PRP, sialic acid and NMN, and from top to bottom, they correspond to T0 and T20. After 20 hours of cell culture, morphological observations showed that the wound healing rate of cells treated with the composition PRP Plus was significantly higher than that of PRP, NMN and sialic acid. The results suggest that the composition PRP Plus can accelerate the healing speed of wounds.

[0078] Example 4: Study on Inhibition of Melanin Production

[0079] The selected Melan-A is a normal mouse-derived melanocyte that can express normal melanin production and is more suitable for research on inhibiting melanin production than B16 cells. In this process, tyrosinase is the rate-limiting enzyme. The absorbance is measured at 405nm using a colorimetric method to detect the content of melanin production in Melan-A cells, which can indirectly detect the activity of inhibiting tyrosinase and reducing melanin production in different samples. The lower the melanin content, the stronger the whitening effect of the sample.

[0080] The specific experimental steps are as follows:

[0081] Melan-A cell line (gift from the University of Hong Kong) was cultured in a CO2 incubator (5% CO2, 37°C) and cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin for 24 hours. Melan-A cells were cultured to a cell concentration of 5×10 5 / mL suspension and inoculated on a 6-well plate. Place in a CO2 incubator (5% CO2, 37°C) and culture for 24 hours. Then add different samples (1% PRP Plus composition in different proportions, 1% PRP, 1mM NMN and 1mM sialic acid) respectively, continue to culture for 24 hours, scrape the cells and transfer them to a 1.5ml centrifuge tube, centrifuge, and centrifuge at a speed of 1000rpm. Aspirate the culture medium, add 1mol / L NaOH for dissolution, and measure the absorbance at 405nm. 10μM H-89 is the positive control, and the blank without drug addition is used as the negative control. The relative content of melanin is 100%. H-89, purchased from Shanghai Yuanye Biotechnology Co., Ltd., is a highly efficient and selective PKA inhibitor that can inhibit melanin formation.

[0082] The experimental results are as follows Figure 3 , the inhibitory effect of different ratios of PRP Plus compositions on melanin formation was compared. Compared with the control group, when PRP:NANA (sialic acid):NMN (3000:1:1) (V / W / W) significantly inhibited melanin formation (P < 0.01), it was the optimal ratio of the composition PRP Plus. PRP plus (100-10000:1:1) all had the effect of inhibiting melanin formation. Sialic acid (NANA) also showed a significant inhibitory effect on melanin formation, indicating that the addition of sialic acid to the PRP plus combination significantly improved the inhibitory effect on melanin and was beneficial for lightening wounds.

[0083] Example 5: Promoting the expression of collagen COLIA1 mRNA

[0084] This study selected Mouse dermal fibroblast cells (primary cultured mouse dermal fibroblasts, MDF), used real-time PCR, and used fluorescent dye luminescence detection to quantify the expression of the target gene COLIA1 mRNA in the sample, and compared the efficacy of 1% PRP plus composition, 1% PRP, 1mM NMN and 1mM sialic acid samples in promoting collagen gene expression. TGF-β1 (transforming growth factor β1) was used as a positive control.

[0085] The specific experimental steps are as follows:

[0086] 1) Primary culture of mouse dermal fibroblasts (MDF)

[0087] Select newborn C57BL / 6 healthy mice, disinfect with alcohol, and cut the back skin with scissors, trying not to cut the subcutaneous fat and connective tissue. Soak the removed skin tissue in Dispase II (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and digest it overnight at 4°C. Take it out the next day and cut the skin into pieces until the tissue is invisible to the naked eye, transfer it to 0.25% trypsin solution, digest and culture it in a 37°C water bath, filter it, and centrifuge it. Resuspend the cells in DMEM culture medium containing 20% ​​FBS, inoculate the suspension in a 100mm culture dish, and culture it in a CO2 incubator (5% CO2, 37°C) for 48 hours. MDF cells are cultured with DMEM culture medium containing 10% FBS and 1% penicillin / streptomycin to a cell concentration of 2×10 4 / mL suspension was inoculated on a 6-well plate and cultured in a CO2 incubator (5% CO2, 37°C) for 24 hours, and then PRP plus, PRP, sialic acid and NMN samples were added. Nothing was added as a negative control, TGF-β1 was added as a positive control, and mRNA was extracted from MDF cells after further culture for 24 hours.

[0088] 2) Total RNA extraction from MDF cells

[0089] After each sample was treated with MDF cells for 24 hours, the culture medium was removed and used RNA was extracted using RT RNA isolation reagent and RNA concentration was measured using Nanodrop. RNA quality and quantity were determined by measuring A260 / A280 and A260 / A230 (ratios greater than 1.8).

[0090] (3) Design the primer sequences as follows:

[0091] COLIA1 F forward primer (SEQ ID NO: 1): 5'-TCT GCG ACA ACG GCA AGG TG-3'

[0092] COL1A1 R reverse primer (SEQ ID NO: 2): 5'-GAC GCC GGT GGT TTC TTG GT-3'

[0093] (4) Real time PCR

[0094] The extracted total RNA samples were normalized to 2 μg and any contaminating genomic DNA was removed using DNase I. 500 ng of RNA samples were reverse transcribed into cDNA according to the experimental manual. Relative gene quantification was performed at 480. Amplification was performed for 45 cycles: each cycle consisted of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 20 seconds. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference and the expression of 2- ΔΔ The mRNA level was determined by Ct value. Figure 4 shown.

[0095] When the skin is injured, most of the time it will stimulate the dermal cells to secrete collagen to rebuild the skin structure. Figure 4 It can be seen that the composition PRP Plus has a significant effect on promoting the expression of collagen COLIA1 mRNA (compared with the control group: *P < 0.05), which is beneficial to skin wound healing; the positive control TGF-β1 **P < 0.01. There is no significant difference among PRP, NMN and sialic acid.

[0096] Example 6: Inhibition of inflammatory factors

[0097] When the skin is injured, skin cells release various cytokines, leading to skin inflammation. This experiment uses the cell model of lipopolysaccharide (LPS)-induced human keratinocytes (HaCaT) inflammation to study and compare the inhibitory effects of PRP, the composition PRP Plus, NMN and sialic acid on inflammation. Human skin keratinocytes (HaCaT) cells were used to pretreat the cells with samples for 3 hours; then real-time PCR was performed to detect that after treating HaCaT cells with the sample prepared in Example 1, LPS (100ng / ml) was added to the cells to induce inflammation, and real-time PCR was used to detect and compare the mRNA expression levels of inflammatory factors IL-6, TNF-α and COX-2 secreted by HaCaT cells.

[0098] The specific experimental steps are as follows:

[0099] 1) HaCaT cells were prepared to a cell density of 3×10 5 / mL of suspension and inoculated on a 12-well plate. Place in a CO2 incubator (5% CO2, 37°C) and culture for 24 hours, then add 1% composition PRP Plus, 1% PRP, 1mM sialic acid and 1mM NMN samples respectively. After pretreatment of the cells for 3 hours, lipopolysaccharide (LPS, 100ng / mL) was added to induce an inflammatory response. The culture medium was used as a blank control, and dexamethasone (10μM) was used as a positive control (Dex). After 24 hours of culture, mRNA was extracted from HaCaT cells. Each group of samples was cultured in 3 replicate wells, and the experiment was repeated 3 times.

[0100] 2) Total RNA extraction: Use Total RNA was extracted from RT RNA and DNA was removed using DNase I. RNA quality and quantity were determined by NanoDrop measurement of the absorbance ratios at A260 / A280 and A260 / A230 (ratios greater than 1.8). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference to standardize the content of total RNA samples in the extract.

[0101] 3) The qRT-PCR primer design sequences of different factors are as follows:

[0102] IL-6F-Forward Primer (SEQ ID NO: 3): 5'-GAGAGTAGTGAGGAACAAGCCAGAGC-3

[0103] IL-6R-reverse primer (SEQ ID NO: 4): 5'-CTA CAT TTG CCG AAG AGC CCT CAG G-3'

[0104] The results are as follows Figure 5 .

[0105] TNF-αF-Forward Primer (SEQ ID NO: 5): 5'-ATG AGC ACT GAA AGC ATG ATC CGG-3'

[0106] TNF-αR-Reverse Primer (SEQ ID NO:6): 5'-GCAATGATCCCAAAGTAGACCTGCCC-3'

[0107] The results are as follows Figure 6 .

[0108] COX-2F-Forward Primer (SEQ ID NO:7): 5'-CTG CGA CTC CTT GAC GTT GA-3'

[0109] COX-2R-reverse primer (SEQ ID NO: 8): 5'-GGT CGT GTA GCG GTG AAA GT-3'

[0110] The results are as follows Figure 7 .

[0111] GAPDH F-Forward Primer (SEQ ID NO:9): 5'-ACA ACT TTG GTA TCG TGG AAG G-3'

[0112] GAPDH R-Reverse Primer (SEQ ID NO: 10): 5'-GCC ATC ACG CCA CAG TTT C-3'

[0113] 4)qRT-PCR (Real time PCR)

[0114] According to the experimental manual, 500 ng of total RNA sample was reverse transcribed into cDNA. Relative gene quantification was performed at 480. PCR amplification was performed for 45 cycles: each cycle consisted of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 20 seconds. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference and was calculated by ΔΔ The Ct value was used to determine the mRNA level.

[0115] Depend on Figure 5 The results showed that PRP plus was significantly better than PRP (P < 0.05) and NMN in inhibiting the inflammatory factor IL-6; sialic acid showed an inhibitory effect on the inflammatory factor IL-6 comparable to the positive control dexamethasone. In the combination of PRP plus, it significantly contributed to its anti-inflammatory effect.

[0116] Depend on Figure 6 The results showed that the PRP plus group and the compound had a significantly better effect in inhibiting the inflammatory factor TNF-α than PRP and NMN, and the anti-inflammatory effect of sialic acid and the positive control was significant (P < 0.001).

[0117] Depend on Figure 7 The results showed that PRP plus inhibited the inflammatory factor COX-2, P < 0.01, which was significantly better than PRP and NMN. Sialic acid and positive control had significant anti-inflammatory effects (P < 0.001).

[0118] By comparing the anti-inflammatory effects of PRP plus, PRP, sialic acid and NMN on inflammatory factors IL-6, TNF-α and COX-2, it was found that the anti-inflammatory effect of PRP plus, a combination of NMN and sialic acid, was significantly increased compared to PRP.

[0119] Example 7: Determination of Nitric Oxide NO Content

[0120] iNOS is an inducible nitric oxide synthase, which is a catalytic enzyme for the production of NOS reactive nitrogen free radicals in the body. Because nitric oxide (NO) is an indicator of inflammation in the body, and because nitric oxide is very unstable, it is easier to detect the content of iNOS, which is an enzyme that produces nitric oxide. Its content may be an indicator of the degree of inflammation in the body. Lipopolysaccharide (LPS) can induce an increase in the level of iNOS in the body or cells, and the addition of the positive control dexamethasone (Dex) can inhibit the level of iNOS by 40%.

[0121] After treating HaCaT skin keratinocytes as in Example 6, the supernatant of HaCaT cells was collected and the NO release was detected according to the iNOS detection kit (nitric oxide (NO) detection kit (lot number GR3311102-1) purchased from Abcam). The results are as follows: Figure 8 .

[0122] Depend on Figure 8 The results showed that compared with the components in the composition, the composition PRP plus can significantly inhibit the level of iNOS and reduce the release of nitric oxide NO, thereby showing its anti-inflammatory effect. By inhibiting the release of nitric oxide NO, it helps prevent infection and accelerate wound healing. Compared with the control group, P < 0.001, it is significantly better than PRP, NMN and sialic acid. It is similar to the positive control dexamethasone (Dex).

[0123] Example 8: Antioxidant Effect of PRP plus

[0124] Bacterial infection of skin wounds has always been a major problem in the wound healing process. Reactive oxygen species (ROS) produced by wounds or bacterial infections will further hinder wound healing, especially seriously affecting wound healing in diabetic patients.

[0125] This study selected a kit (Total Antioxidant Capacity (TAC) Colorimetric Assay Kit, K274-100, purchased from Biovision) for detection, and used Trolox (tocopherol acetate: a derivative of vitamin E) to establish a standard curve for its ability to resist copper ion oxidation, and calculated the total antioxidant capacity (expressed as the equivalent concentration of Trolox) of different proportions of 1% composition PRPPlus, 1% PRP, 1mM sialic acid and 1mM NMN) based on the standard curve. In this test, the higher the equivalent concentration with Trolox, the better the antioxidant capacity of the sample.

[0126] The results are as follows Fig. 9 Different proportions of PRP plus all showed antioxidant effects, among which PRP:NANA (sialic acid):NMN (3000:1:1) (V / W / W) was significantly better than other groups such as PRP and sialic acid (P < 0.001) and NMN (P < 0.05). Therefore, the composite PRP plus can significantly enhance the antioxidant effect of PRP, reduce the level of ROS and promote wound healing.

[0127] Example 9: Wound healing experiment with blood from different animal sources

[0128] Fresh blood from cattle, horses, sheep, pigs, deer, chickens, and rabbits was taken, and PRP plus samples of cattle, horses, sheep, pigs, deer, chickens, and rabbits were prepared according to the method of Example 1, and wound healing experiments were carried out according to Example 3. The results are as follows: Fig.10 . It shows that the blood PRP plus samples of other animals have a certain effect in promoting wound healing. In comparison, the effect of humans and deer is better.

[0129] Example 10: Experiment on promoting wound healing in mice

[0130] Twelve healthy C57BL / 6 mice without specific pathogens, aged 6 weeks and weighing 20-25 g, were purchased from the Animal and Plant Care Center of the Hong Kong University of Science and Technology and were kept in a 12-hour light / 12-hour dark environment. The laboratory temperature was controlled at (25±1)℃, the relative humidity was 50%-70%, and food and water were freely available. The experiment began after the animals adapted to the environment for 1 week. The use and operation of experimental animals in the study complied with the requirements of the Hong Kong University of Science and Technology on experimental animal operation and animal ethics.

[0131] Experimental steps: The mice were anesthetized with chloral hydrate, and after shaving the back, a hole was punched on the left and right sides of the mouse's back with a 0.5 cm diameter puncher. The left side was added with saline as a control, and the right side was added with 1% PRP plus or 1% PRP (v / v, the solvent was saline). The drug was added once a day.

[0132] The results are as follows Fig.11 It can be seen that after 6 days of treatment with PRP plus and PRP, compared with normal saline, PRP plus has a significant effect on promoting wound healing in mice and is superior to PRP.

[0133] Example 11: Promoting wound healing

[0134] This example is about a 2-year-old child who was accidentally scalded during the Spring Festival of 2021. After seven days of medical treatment, the wound was still leaking and inflamed. The child's parents voluntarily tried PRP plus (concentration of 1% (v / v), solvent is normal saline), and the results are as follows: Fig.12As shown in the figure, PRP plus had anti-inflammatory effect and good scab formation on the third day after application; the wound continued to heal itself four days after discontinuation of application.

[0135] Example 12: Improving scars and reducing itching

[0136] This example is about a police officer who was burned by a strong water shower while patrolling. He went to the doctor for treatment. Although the wound healed, the pain and itching were unbearable. He voluntarily used PRP plus (concentration of 1% (v / v), the solvent was normal saline), and the scar thickness of the wound was reduced and the itching was improved. The results are as follows: Fig.13 shown.

[0137] Example 13: Accelerating the healing of diabetic foot wounds

[0138] Case 1: In 2023, with the consent of the diabetic patient and the doctor, the patient voluntarily tried PRP plus (concentration of 1% (v / v), solvent was saline). After 7 days of trial, the wound scabped and the results were as follows: Fig.14 shown.

[0139] Case 2: In 2023, with the consent of the diabetic patient and the doctor, the patient voluntarily tried PRP plus (concentration 1% (v / v), solvent was saline). After 16 days of trial, the wound scabped. The results were as follows: Fig.15 shown.

Claims

1. A composition capable of resisting inflammation and promoting skin repair, characterized in that: The composition comprises, in addition to platelet-rich plasma, β-nicotinamide mononucleotide and sialic acid.

2. The composition according to claim 1, characterized in that The volume-to-mass ratio of platelet-rich plasma, sialic acid and β-nicotinamide mononucleotide in the composition is (100-10000):1:1 (V / W / W).

3. The composition according to claim 2, characterized in that The volume-to-mass ratio of platelet-rich plasma, sialic acid and β-nicotinamide mononucleotide in the composition is (1000-5000):1:1 (V / W / W); Preferably, the volume-to-mass ratio of platelet-rich plasma, sialic acid and β-nicotinamide mononucleotide in the composition is (2500-3500):1:1 (V / W / W); More preferably, the volume-to-mass ratio of platelet-rich plasma, sialic acid and β-nicotinamide mononucleotide in the composition is 3000:1:1 (V / W / W).

4. The composition according to any one of claims 1 to 3, characterized in that The platelet-rich plasma is derived from humans, deer, cattle, horses, sheep, pigs, chickens, rabbits or fish.

5. The method for preparing the composition according to any one of claims 1 to 4, characterized in that: The preparation method comprises uniformly mixing platelet-rich plasma, β-nicotinamide mononucleotide and sialic acid to prepare the composition; Preferably, the uniform mixing is performed by ultrasonication at 90 to 110 W for 8 to 12 minutes, preferably at 100 W for 10 minutes.

6. The preparation method according to claim 5, characterized in that: The platelet-rich plasma is obtained by centrifugation from fresh blood isolated from the body; Preferably, the centrifugal separation is differential centrifugation, which includes centrifuging the fresh blood at 1200-2000 rpm for 4-6 minutes and then at 3000-3800 rpm for 18-22 minutes; More preferably, the differential centrifugation comprises centrifuging the fresh blood at 1500 rpm for 5 minutes and then at 3500 rpm for 20 minutes.

7. The preparation method according to claim 5 or 6, characterized in that: The preparation method further comprises freeze-drying the uniformly mixed mixture to obtain the composition; Preferably, the freeze-drying temperature is -80°C.

8. Use of the composition according to any one of claims 1 to 4 in the preparation of skin repair medicines or cosmetics.

9. The use according to claim 8, characterized in that The skin repair includes one or more selected from skin regeneration, skin anti-aging and assisting skin wound healing; And / or, the concentration of the composition in the medicine or cosmetic is 0.1-5.0% (V / V), preferably 0.5-1.5% (V / V).

10. The use according to claim 9, characterized in that The skin trauma includes one or more selected from skin scald, skin burn, atopic dermatitis and diabetic foot, and the skin anti-aging includes skin anti-oxidation; And / or, the concentration of the composition in the medicine or cosmetic is 1.0% (V / V), for example, the composition is adjusted to the concentration using physiological saline.

Images