CN105131086B - A kind of hexapeptide and its application - Google Patents
A kind of hexapeptide and its application Download PDFInfo
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- CN105131086B CN105131086B CN201510631206.9A CN201510631206A CN105131086B CN 105131086 B CN105131086 B CN 105131086B CN 201510631206 A CN201510631206 A CN 201510631206A CN 105131086 B CN105131086 B CN 105131086B
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Abstract
The invention discloses a kind of hexapeptide and its applications.The amino acid sequence of the synthesis hexapeptide is as follows：Gly Met Cys Cys Ser Arg, are abbreviated as GMCCSR, molecular weight 654.0.The polypeptide of the present invention uses Peptide synthesizer, is synthesized using solid-phase synthesis.The object of the present invention is to provide a kind of with antioxidation activity in vitro, protection erythrocyte hemolysis and the synthesis hexapeptide for promoting the proliferation of human skin fibroblasts and collagen to generate, and can be applied to the fields such as bio-pharmaceuticals and cosmetics.
Description
Technical Field
The invention relates to the fields of biological pharmacy, cosmetics and the like, in particular to synthetic hexapeptide and application thereof.
Background
The bioactive peptide has various human metabolism and physiological regulation functions, is easy to digest and absorb, has the functions of promoting immunoregulation, resisting bacteria, reducing blood pressure, resisting cancer, resisting oxidation and the like, and is formed by 25 natural amino acids in different compositions and arrangement modes from dipeptide to different peptides with complex linear and annular structures through peptide bonds. The active peptide has extremely high safety and is the hottest research topic and the functional factor with great development prospect in the current international food industry. Modern nutrition shows that protein is not completely absorbed in the form of free amino acid after being acted by digestive tract enzyme, and is mostly absorbed in the form of low peptide, and the low peptide has higher nutritional value and biological value than the free amino acid.
Since the middle of the twentieth century, with the improvement of living standard and the rapid development of subjects such as cell biology and molecular biology, scientists have advanced the research on aging mechanism, wherein the widely studied theories include the theory of free radical-oxidative stress, the theory of inflammation, the theory of immunity, the theory of mitochondrial DNA, and the like, and the theory of free radical-oxidative stress is most important and can be linked with other theories to play a role. The development of the theory was initiated by Harman in 1956, who thought that free radicals were the main culprit of truly induced aging of the body and the key to various malignant diseases such as malignant hyperplasia of body tissues. In 1990, professor Sohal in the united states developed the theory, the concept of oxidative stress was first proposed, and developed on the basis of the theory of free radicals to form the theory of free radical-oxidative stress. Specifically, the normal body establishes itself and forms a complete set of defense systems to avoid the damage and destruction of free radicals, such as various antioxidant enzymes present in cells, such as Superoxide Dismutase, SOD; glutathieone Peroxidase, GSH-Px and the like, the capability of organism to eliminate oxygen free radical is directly related to the activity of the organism. Once the organism can not remove the free radicals in time due to excessive increase of the free radicals caused by internal or external reasons, the free radicals carry unpaired single electrons and can attack cells in the body, unsaturated fatty acids of biological membranes can be oxidized, protein cross-linking denaturation can be induced, even cell nucleuses and mitochondrial DNA or RNA and the like can be damaged, so that the cells and tissues are damaged, and finally, the loss of the functions of the organism and the occurrence of diseases related to aging are caused. Thus, the elimination of excess free radicals in the body plays an important role in the process of preventing certain diseases.
The ability of a sample to scavenge free radicals is the most common method for assessing its antioxidant activity. ABTS, 2, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, a water-soluble substance which can be MnO2The antioxidant can provide hydrogen atoms to reduce the free radicals into colorless ABTS, and the ABTS scavenging by the antioxidant can be evaluated according to the change of the absorbance of the solution at 736 nm+The ability of the cell to perform. DPPH (1, 1-diphenyl-2-pichydrazino) is an alcohol-soluble substance that forms nitrogen in an alcohol solutionThe free radical has a maximum absorption peak at 522 nm, and after the antioxidant sample is added, the solution fades, and the antioxidant activity of the sample can be detected through the change of the light absorption value of the solution. The FRAP method (ferrreducing antioxidant potential Assay) is a method for measuring the antioxidant capacity of a substance. The principle is that under the acidic condition, the antioxidant can reduce ferri-tripyridyltriazine (Fe)3+TPTZ) produces Fe in blue2+TPTZ, followed by determination of blue Fe at 596 nm2+TPTZ gives the total antioxidant capacity in the sample. The three models are widely applied due to simple and convenient operation, rapidness and good controllability.
The construction of a cell oxidative damage model is a common method for evaluating whether a sample has antioxidant capacity, and red blood cells are common materials in vitro biological experiments due to rich sources and convenient material acquisition. Free radicals first attack lipids and proteins on the red blood cell membrane, causing the membrane to break, releasing hemoglobin from the red blood cells, known as red blood cell hemolysis. AAPH, H2O2And Hemin is a commonly used substance inducing hemolysis of red blood cells. The sample scavenges free radicals present in the system to inhibit hemolysis of the red blood cells. An Atomic Force Microscope (AFM) is a novel imaging tool with high resolution, and has been widely used in characterization of cell morphology and ultrastructure thereof in the biomedical field. Free radicals attack cell membranes causing their loss of structure and function, leading to cellular hemolysis. This conclusion was further verified by observing the morphology of the red blood cells in the control, injured and protected groups using Atomic Force Microscopy (AFM).
Skin aging is primarily a result of a person's genetic predisposition (also known as chronological aging) and the person's physiological response to environmental stress (also known as photoaging). Photoaging is the effect of the biological response of the external environment to the skin and is a widely applicable model of aging experiments. The skin's response to photoaging is often associated with a lack of normal hydration, skin sagging, and the appearance of lines and wrinkles. UVB irradiation can accumulate Reactive Oxygen Species (ROS) in fibroblasts, exceed the scavenging capacity of the ROS, break the balance of oxidation and antioxidation, generate oxidative stress reaction, regulate a series of programmed cell reactions, even regulate and control signal paths related to senescence, and promote the generation of cell senescence. Research shows that the accumulation of ROS in cells is an important link in the development process of skin photoaging. Human skin fibroblasts are the main sites for collagen production in human skin, and the quantity and yield of cellular collagen are closely related to the aging state of skin. Therefore, the survival rate of human skin fibroblasts and the yield of collagen are widely recognized indicators for evaluating the aging state of skin.
Disclosure of Invention
The invention aims to provide a synthetic hexapeptide which has in vitro antioxidant activity, protects erythrocyte hemolysis, promotes the proliferation of human skin fibroblasts and the generation of collagen, and can be applied to the fields of biological pharmacy, cosmetics and the like.
The synthetic hexapeptide of the invention is abbreviated as GMCCSR, the molecular weight is 654.0, and the sequence is as follows: Gly-Met-Cys-Cys-Ser-Arg. Wherein,
gly represents the corresponding residue of amino acid with English name of Glycine and Chinese name of Glycine;
met represents the corresponding residue of an amino acid having the english name Methionine and the chinese name Methionine;
cys represents the corresponding residue of an amino acid with the English name Cystein and the Chinese name Cysteine;
ser represents the corresponding residue of an amino acid with the english name Serine and the chinese name Serine;
arg represents the corresponding residue of the amino acid known by the English name Arginine and the Chinese name Arginine.
The amino acid sequence of the invention adopts a standard Fmoc scheme, and a reasonable polypeptide synthesis method is realized by screening resin. The C-terminal carboxyl group of the target polypeptide is covalently linked to an insoluble polymeric resin, and then the amino group of the amino acid is used as a starting point to react with the carboxyl group of another molecule of amino acid to form a peptide bond. The process is repeated continuously to obtain the target polypeptide product. And after the synthesis reaction is finished, removing the protecting group, and separating the peptide chain from the resin to obtain the target product. Polypeptide synthesis is a process of repeated addition of amino acids, and the solid phase synthesis sequence is synthesized from the C-terminus to the N-terminus.
According to the invention, the hexapeptide with the concentration of 1-100 mug/mL is mixed with the red blood cells, and the hemolysis rate of the red blood cells is reduced after incubation for 2 h under the action of AAPH with the final concentration of 100 mM, and the appearance of the hexapeptide is smoother and ordered than that of a pure AAPH treatment group, and is in a cake-shaped structure. The inhibition rate of the cell hemolysis reaches 91.80 +/-1.34%.
Further, the hexapeptide has a concentration of 5 mug/mL, and the hexapeptide enables the SOD activity of the red blood cells to be increased from 2.5976mgprot/mL to 6.4819 mgprot/mL.
Further, under the action of UVB, the hexapeptide is mixed with human skin fibroblasts, and after incubation for 72 hours, the cell survival rate is improved by 7.5% -19.1%; meanwhile, the yield of the collagen is improved by 3.27% -16.53%, and the concentration of the hexapeptide is 1-10 mug/mL.
In further implementation, the synthetic hexapeptide is uniformly mixed with normal human red blood cells, the mixture is incubated, after AAPH damage is carried out for 2 hours, the inhibition rate of cell hemolysis reaches 91.80 +/-1.34%, and compared with an erythrocyte group only damaged by AAPH, the appearance of the erythrocyte group treated by the synthetic hexapeptide of 100 microgram/mL is smoother and ordered than that of a pure AAPH treatment group, and is in a cake-shaped structure. After the treatment of the synthetic hexapeptide with the concentration of 5 mug/mL, the SOD activity in the cells is increased from 2.5976mgprot/mL to 6.4819 mgprot/mL, and the result shows that the SOD activity of the red blood cells treated by the synthetic hexapeptide is greatly improved, and the synthetic hexapeptide can be applied to the fields of biological medicine, cosmetic preparation and the like.
Go toStep by step, the invention is carried out in UVB (80 mJ/cm)2) Adding the synthetic hexapeptide with the concentration of 1-10 mug/mL into the human skin fibroblast culture solution for incubation for 72 hours, so that the cell survival rate is improved by 7.5% -19.1%; meanwhile, the yield of the collagen is improved by 3.27 to 16.53 percent. The collagen yield is increased from 11.9512 +/-1.2067 mug/mL of the model group to 13.9268 +/-1.3409 mug/mL of the synthesized hexapeptide group.
Compared with the prior art, the invention has the following advantages and technical effects:
the invention synthesizes the peptide for the first time, and adopts an in vitro chemical antioxidant method to detect the antioxidant activity of the synthesized hexapeptide, and the synthesized hexapeptide has strong DPPH and ABTS removing effect+The compound hexapeptide has the advantages of having strong free radical capacity and electron supply capacity, being applied to protecting normal human erythrocytes with AAPH damage, obviously reducing the hemolysis rate of erythrocytes, greatly improving the SOD activity, promoting the growth of human skin fibroblasts with ultraviolet damage by synthesizing hexapeptide, and promoting the generation of collagen.
Drawings
FIG. 1 is an ESI-MS spectrum of the synthesis of the hexapeptide Gly-Met-Cys-Cys-Ser-Arg with m/z (mass to charge ratio) on the abscissa and intensity on the ordinate.
FIG. 2 is a graph comparing the ABTS free radical scavenging activity of the synthetic hexapeptide Gly-Met-Cys-Cys-Ser-Arg.
FIG. 3 is a graph comparing the DPPH free radical scavenging activity of the synthetic hexapeptide Gly-Met-Cys-Cys-Ser-Arg.
FIG. 4 is a diagram showing the reduction capacity for the synthesis of the hexapeptide Gly-Met-Cys-Cys-Ser-Arg.
FIG. 5 is a graph showing the change in the inhibition of hemolysis of normal human erythrocytes after AAPH damage after the addition of synthetic hexapeptide.
FIGS. 6 a-6 c are Atomic Force Microscope (AFM) images of different treatment groups of red blood cells.
Detailed Description
The present invention is further illustrated by the following specific examples, but the scope of the invention is not limited thereto. For process parameters not specifically noted, reference may be made to conventional techniques.
The invention evaluates the antioxidant and anti-skin aging activities of the polypeptide by ABTS free radicals, DPPH free radical scavenging experiments, FRAP experiments, APPH induced erythrocyte hemolysis experiments and ultraviolet injury human skin fibroblasts.
Solid phase synthesis of polypeptides
Selecting high molecular resin (Shanghai Jie peptide Biotech Co., Ltd.), connecting the carboxyl of Arg with a resin in a covalent bond form according to the characteristics of an amino acid sequence Gly-Met-Cys-Ser-Arg, then carrying out a shrinkage reaction on the amino of Arg and the carboxyl of Ser, adding the amino of Cys and Ser to react with the carboxyl of Cys after treatment, sequentially adding amino acid from right to left, adding the last Gly amino acid, and then cutting off the resin to obtain the target polypeptide. Purifying by high performance liquid chromatography to obtain final product, wherein kromasil C is used in the purification process18-5 (4.6 x 250 mm) chromatography column with flow rate of 1.0 mL/min. The liquid phase system uses two channels, solvent A is acetonitrile containing 0.1% trifluoroacetic acid; solvent B-water containing 0.1% trifluoroacetic acid. The gradient elution was as follows: the initial proportion of A is 7%, the proportion of A is increased to 32% within 0.01min to 25min, the proportion of A is increased to 100% within 25min to 25.1min, the operation is kept at 100% for 30min, and the detection wavelength is 220 nm. The polypeptide solution was collected, snap-cooled with liquid nitrogen, and then lyophilized. The product was obtained with a purity of 95% or more and the structure was identified by ESI-MS (as shown in FIG. 1).
DPPH free radical scavenging activity experiment for synthesizing hexapeptide
DPPH experiment: mu.L of a sample with a mixed concentration of 1-100. mu.g/mL of 180. mu.L of DPPH reagent (150. mu.M DPPH, solvent 80% ethanol, v/v) was added to a 96-well plate. A blank set of 180. mu.L of 80% ethanol mixed with 20. mu.L of ultrapure water and a model set of 180. mu.L of DPPH reagent mixed with 20. mu.L of ultrapure water were set simultaneously. Measuring the absorbance value at 522 nm by using a microplate reader (infiniteM 200 pro, TECAN, Switzerland), setting a program, shaking for 30 s, measuring once every 2min for 20 periods, wherein the absorbance value of the model group is adjusted to be between 0.7 and 0.8 before the experiment, and keeping the room temperature and avoiding light in the measuring process. The results are shown in FIG. 2, where a is the kinetic profile of ABTS free radical scavenging for samples of different concentrations within 40 min; and b is the significance analysis of the effect of the sample among different concentrations when the sample acts for 40 min. In fig. 2 a, the abscissa is Time and the ordinate is ABTS scavenging rate. In FIG. 2, b has a Concentration on the abscissa and an ABTS scavenging rate (ABTS radical scavenging rate) on the ordinate.
The result shows that the peptide has obvious effect of eliminating ABTS free radicals, basically achieves the maximum effect of eliminating ABTS free radicals within 2min after the peptide is added into a sample, and the peptide has IC (integrated Circuit) for eliminating ABTS free radicals50The value was 16.94 μ M, which was lower than 26.18 μ M of the positive control vitamin C.
ABTS free radical scavenging activity experiment for hexapeptide synthesis
ABTS experiment: mixing 180 mu L of ABTS reaction liquid with 20 mu L of sample of 1-100 mu g/mL, adding the mixture into a 96-well plate, shaking and uniformly mixing. A blank set of 180. mu.L of 80% ethanol mixed with 20. mu.L of ultrapure water and a model set of 180. mu.L of DPPH reagent mixed with 20. mu.L of ultrapure water were set simultaneously. The absorbance at 736 nm was determined with a microplate reader (infinite M200 pro, TECAN, Switzerland). Setting a program, shaking for 30 s, measuring once every 2min, and measuring for 20 periods in total, wherein before the experiment, the absorbance value of the model group is adjusted to be between 0.7 and 0.8, and the room temperature and the light are kept away in the measuring process. FIG. 3 shows DPPH free radical scavenging activity of the synthetic hexapeptide Gly-Met-Cys-Cys-Ser-Arg. a is a scavenging kinetic curve of samples with different concentrations to DPPH free radicals within 40 min; and b is the significance analysis of the effect of the sample among different concentrations when the sample acts for 40 min. Where the abscissa of a in fig. 3 is Time and the ordinate is DPPH scavenging rate. In FIG. 3, b has a Concentration on the abscissa and a DPPH scavenging rate on the ordinate.
The results show that the peptide has more remarkable scavenging effect on DPPH free radicals and IC50The value was 36.93 μ M, slightly higher than 27.42 μ M for the positive control vitamin C.
FRAP experiment for hexapeptide synthesis
FRAP experiment: 180 uL FRAP reaction solution (V)300 mM acetic acid buffer: V20 mM TPTZ: V20 mM ferric chloride= 10:1: 1), mixing 20 μ L of 1-100 μ g/mL sample, adding into a 96-well plate, shaking, and mixing uniformly. A blank group of 200. mu.L of ultrapure water and a model group of 180. mu.L of FRAP reaction solution mixed with 20. mu.L of ultrapure water were set simultaneously. Absorbance at 596 nm was measured with a microplate reader (infiniteM 200 pro, TECAN, Switzerland), programmed, shaken for 30 s, every 2min for a total of 20 cycles.
FIG. 4 is a diagram showing the reduction capacity for the synthesis of the hexapeptide Gly-Met-Cys-Cys-Ser-Arg. a is samples with different concentrations within 40min3+Reduction to TPTZ-Fe2+The kinetic curve of (a); and b is the significance analysis of the effect of the sample among different concentrations when the sample acts for 40 min. In fig. 4, the abscissa of a is Time and the ordinate is FRAP value (antioxidant activity). In FIG. 4, the abscissa of b represents Concentration and the ordinate represents FRAP value (antioxidant activity).
The result shows that the synthesized hexapeptide has certain reducing capacity, and the reducing capacity of 152.49 mu M is equivalent to that of 9.82 mu M vitamin C.
Application of synthesized hexapeptide in protecting UVB damage of human skin fibroblasts
Human skin fibroblasts were cultured in complete medium consisting of basal medium high-glucose DMEM, 10% fetal bovine serum (v/v), and 1% diabody (consisting of penicillin and streptomycin, v/v).Placing at 37 ℃ in CO2A saturated humidity incubator with volume fraction of 5%. Change the solution 1 time every 2 days. Inoculating the cells to 96-well culture plate with 100 μ L/well and concentration of 5 × 10 when the cells are about 90% of the culture flask4cells/mL. After the cells had grown for 24 h, the culture was aspirated, washed 1 time with 200. mu.L PBS, 200. mu.L PBS was added, and 80mJ/cm of the solution was used2UVB irradiation, PBS aspiration, 200. mu.L of sample (zero concentration replaced by complete medium, which is normal control) per well, culture for 72 h, aspiration of culture solution, and cell survival rate detection by MTT method, the results are shown in Table 1.
TABLE 1
| UVB | Synthesis of hexapeptide + UVB | |
| Human skin fibroblast survival rate | 1 | 1.191 |
Application of synthetic hexapeptide in promoting generation of UVB-damaged human skin fibroblast collagen
Collecting the cells of the above treated group, removing the culture solution, washing the cell surface with sterile water twice, adding 200 μ L ice-cold 70% ethanol, fixing at-80%oPlacing in refrigerator for at least 10min, taking out, washing cell surface with sterile water twice, air drying, adding 200 μ L1% (m/v) saturated picric acid-scarlet dye solution into each well, and standing at 4%oShaking gently for 24 h under C condition, removing the dye solution, washing with sterile water for 3 times until no dye solution is washed out, adding 150 μL1M NaOH solution at 150r/min, shaking for 15min, taking 100 μ L of each well, placing in a 96-well plate, and measuring the absorbance value at 492nm, wherein the results are shown in Table 2.
TABLE 2
| UVB | Synthesis of hexapeptide + UVB | |
| Human skin fibroblast collagen yield (mug/mL) | 11.9512±1.2067 | 13.9268 ±1.3409 |
Application of synthesized hexapeptide in protection of normal human red blood cell AAPH damage
Blood of healthy adults (under 30 years old) was drawn with an anticoagulation tube containing sodium citrate (anticoagulant sodium citrate: blood = 1: 9, v/v) and stored in a refrigerator at 4 ℃ for use within a week. For use, the blood is placed in a centrifuge tube and centrifuged at 1000-1500 g for 8-15 min, the plasma in the upper layer is removed, and the red blood cells are washed 2-3 times with PBS (pH = 7.4) until the supernatant is colorless. And finally, centrifuging the red blood cells for 8 to 15min at 1000 to 1500g in a centrifuge, removing supernatant to obtain compact hematocrit, and diluting the red blood cells into red blood cell suspension with the concentration of 20% (volume, v/v) by using PBS. The samples were determined to be nonhemolytic by treating erythrocytes with 100. mu.g/mL synthetic hexapeptide alone and incubating with 0.1 mL of 20% erythrocyte suspension and 0.3 mL of PBS for 2 h as a blank control.
Application example 1
0.1 mL of 20% erythrocyte suspension is pretreated for 20 min by 0.1 mL of synthetic hexapeptide (PBS is used for replacing the hexapeptide, namely, the model group) with the concentration of 0 mu g/mL, 0.2 mL of AAPH solution with the final concentration of 100 mM is added, slight vibration and lightproof incubation are carried out for 2 h, 50 mu L of reactant solution of each treatment group is diluted to 1 mL by PBS buffer solution, 1500g of the reactant solution is centrifuged for 12min, supernatant is taken out and put in a 96-well plate, the absorbance of the supernatant is measured at 540 nm by a microplate reader, and similarly, the reaction mixture is diluted by distilled water to be used as a total blood-dissolved control, and the hemolysis rate is calculated, and the result is shown in figure 5. Meanwhile, the cells of each treatment group were centrifuged at 1500g for 12min by a centrifuge, the cell pellet was collected, washed with PBS for 3 times for centrifugation, the supernatant was discarded, and resuspended again. During flaking, 10 μ L of the erythrocyte resuspension solution is uniformly coated on a clean mica sheet (cells are required to be free from overlapping and agglomeration), then 2.5% (volume, v/v) of glutaraldehyde is used for fixing the cells, after 5min, the silicon wafer is washed with ultrapure water for 3 times, naturally air-dried, finally placed on an atomic force microscope, the morphology of the cells is observed by scanning in a tapping mode, experimental data are analyzed by using Nanoscope software, and the result is shown in FIG. 6 b.
Application example 2
0.1 mL of 20% erythrocyte suspension is pretreated for 20 min by 0.1 mL of synthetic hexapeptide with the concentration of 1 mu g/mL, 0.2 mL of AAPH solution with the final concentration of 100 mM is added, the mixture is incubated for 2 h under slight vibration and in the dark, 50 mu L of reactant solution of each treatment group is diluted to 1 mL by PBS buffer solution, 1500g of the reactant solution is centrifuged for 12min, supernatant is taken out and put in a 96-well plate, the absorbance of the supernatant is measured at 540 nm by a microplate reader, and similarly, the reaction mixture is diluted by distilled water to be used as a full blood-soluble control, and the hemolysis rate is calculated, and the result is shown in figure 5.
Application example 3
0.1 mL of 20% erythrocyte suspension is pretreated for 20 min by 0.1 mL of synthetic hexapeptide with the concentration of 5 mu g/mL, 0.2 mL of AAPH solution with the final concentration of 100 mM is added, the mixture is incubated for 2 h under slight vibration and in the dark, 50 mu L of reactant solution of each treatment group is diluted to 1 mL by PBS buffer solution, 1500g of the reactant solution is centrifuged for 12min, supernatant is taken out and put in a 96-well plate, the absorbance of the supernatant is measured at 540 nm by a microplate reader, and similarly, the reaction mixture is diluted by distilled water to be used as a full blood-soluble control, and the hemolysis rate is calculated, and the result is shown in FIG. 5.
FIG. 5 is a graph showing the change in the inhibition of hemolysis of normal human erythrocytes after AAPH damage after the addition of synthetic hexapeptide. Wherein the abscissa is Concentration and the ordinate is Hemolysis inhibition. The results show that the hemolysis inhibition rate of the sample group is remarkably increased compared with the model group. When the sample concentration is 100 mug/mL, the hemolysis inhibition rate of the sample is not significantly different from that of the red blood cells in the normal group.
Application example 4
0.1 mL of 20% erythrocyte suspension is pretreated for 20 min by 0.1 mL of synthetic hexapeptide with the concentration of 100 mu g/mL, 0.2 mL of AAPH solution with the final concentration of 100 mM is added, the mixture is incubated for 2 h under slight vibration and in the dark, 50 mu L of reactant solution of each treatment group is diluted to 1 mL by PBS buffer solution, 1500g of the reactant solution is centrifuged for 12min, supernatant is taken out to be placed in a 96-well plate, the absorbance of the supernatant is measured at 540 nm by a microplate reader, and similarly, the reaction mixture is diluted by distilled water to be used as a full blood-soluble control, and the hemolysis rate is calculated. (results are shown in FIG. 5) at the same time, the cells of each treatment group were centrifuged at 1500g for 12min, the cell pellet was collected, washed with PBS 3 times for centrifugation, the supernatant was discarded, and resuspended again. During flaking, 10 μ L of the erythrocyte resuspension solution is uniformly coated on a clean mica sheet (cells are required to be free of overlapping and agglomeration), then 2.5% of glutaraldehyde is used for fixing the cells, after 5min, the silicon wafer is washed with ultrapure water for 3 times, naturally air-dried, finally placed on an atomic force microscope, the morphology of the cells is observed by scanning in a tapping mode, experimental data are analyzed by using NanoScope software, and the result is shown in FIG. 6 c.
Application example 5
0.1 mL of 20% erythrocyte suspension is pretreated for 20 min by 0.1 mL of synthetic hexapeptide with the concentration of 0 mu g/mL (replaced by PBS, namely a normal control group), 0.2 mL of AAPH solution with the final concentration of 0mM (replaced by PBS, namely a normal control group) is added, slight vibration and dark incubation are carried out for 2 h, 50 mu L of reactant solution of each treatment group is taken and diluted to 1 mL by PBS buffer solution, 1500g of the reactant solution is centrifuged for 12min, supernatant is taken and put in a 96-well plate, the absorbance of the supernatant is measured at 540 nm by a microplate reader, and the reaction mixture is similarly diluted by distilled water as a total blood-dissolved control to calculate the hemolysis rate, and the result is shown in figure 5. Meanwhile, the cells of each treatment group were centrifuged at 1500g for 12min by a centrifuge, the cell pellet was collected, washed with PBS for 3 times for centrifugation, the supernatant was discarded, and resuspended again. During flaking, 10 μ L of the erythrocyte resuspension solution is uniformly coated on a clean mica sheet (cells are required to be free of overlapping and agglomeration), then 2.5% of glutaraldehyde is used for fixing the cells, after 5min, the silicon wafer is washed with ultrapure water for 3 times, naturally air-dried, finally placed on an atomic force microscope, the morphology of the cells is observed by scanning in a tapping mode, experimental data are analyzed by using NanoScope software, and the result is shown in figure 6 a.
FIGS. 6 a-6 c are Atomic Force Microscope (AFM) images of different treatment groups of red blood cells. Wherein a is normal red blood cells, b is red blood cells treated for 2 h by 100 mM AAPH, and c is red blood cells treated for 2 h by 100 mM AAPH after being pretreated for 20 min by 100 mug/mL synthetic hexapeptide Gly-Met-Cys-Cys-Ser-Arg.
The results show that normal erythrocytes have a typical biconcave structure, the cell surface is smooth, and the peripheral height is basically consistent. The cell surface of AAPH treated group becomes rough, the cells collapse seriously, the height is reduced, and the shape is irregular. The protective group pretreated by the synthetic hexapeptide obviously weakens the damage degree of cells and presents a cake-shaped structure.
Application example 6
TABLE 3
| AAPH | Synthesis of hexapeptide + AAPH | |
| SOD activity (mgprot/mL) | 2.5976 | 6.4819 |
Pretreating 0.1 mL of 20% erythrocyte suspension with 0.1 mL of synthetic hexapeptide with concentration of 5 μ g/mL (substituted by PBS, namely a normal control group) for 20 min, adding 0.2 mL of AAPH solution with final concentration of 0mM (substituted by PBS, namely a normal control group), slightly vibrating, incubating in dark for 2 h, centrifuging the cells of each treatment group at 1500g for 12min, collecting cell precipitate, washing with PBS for 3 times, centrifuging, adding 4oC pre-cooled ultrapure water, 150r/min shaking for 10min, 10000g centrifuging for 30min, collecting supernatant for later use, and measuring SOD activity according to the instruction of the kit (Nanjing kit), wherein the results are shown in Table 3.
Claims (4)
1. A hexapeptide is characterized in that the amino acid sequence of the hexapeptide is Gly-Met-Cys-Cys-Ser-Arg.
2. The hexapeptide according to claim 1, characterized in that under the action of AAPH with a final concentration of 100 mM, the hexapeptide with a concentration of 1-100 μ g/mL is mixed with red blood cells, and after incubation for 2 h, the hexapeptide can reduce the hemolysis rate of the red blood cells, and the morphology of the hexapeptide is smoother, ordered and in a cake-like structure than that of a pure AAPH treatment group; the inhibition rate of cell hemolysis reaches 91.80 +/-1.34%.
3. The hexapeptide according to claim 2, wherein the hexapeptide can increase the SOD activity of red blood cells from 2.5976mgprot/mL to 6.4819 mgprot/mL when the hexapeptide is at a concentration of 5 μ g/mL.
4. The hexapeptide according to claim 2, wherein under the action of UVB, when the concentration of the hexapeptide is 1-10 mug/mL, the hexapeptide can be mixed with human skin fibroblasts, and after incubation for 72 hours, the cell survival rate is improved by 7.5% -19.1%; meanwhile, the yield of the collagen is improved by 3.27 to 16.53 percent.
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