CN106749525A - A kind of small peptide and its application - Google Patents
A kind of small peptide and its application Download PDFInfo
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- CN106749525A CN106749525A CN201611116452.1A CN201611116452A CN106749525A CN 106749525 A CN106749525 A CN 106749525A CN 201611116452 A CN201611116452 A CN 201611116452A CN 106749525 A CN106749525 A CN 106749525A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The present invention provides a kind of small peptide and its application, is related to biological technical field.The amino acid sequence of the small peptide such as SEQ ID NO:Shown in 1.The present invention also provides application of the small peptide in functional food, anti-oxidation function product, the free base product of removing, anti-lipid peroxidation product that the functional food for alleviating diabetic complication, antiatherosclerosis are formed is prepared.Small peptide of the present invention, not only there is stronger oxidation resistance, and the apoptosis of smooth muscle cell under the VSMCs abnormality proliferations of high sugar induced, regulation sugar environment high can be adjusted, it is finally set to tend to normal condition, therefore, atherogenesis and alleviate the aspects such as diabetes other complication and have very big DEVELOPMENT PROSPECT in the case where sugar environment high is prevented.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of small peptide and its application.
Background technology
Diabetes are one group and are increased the metabolic disease group being characterized with chronic blood sugar level.The glucose that hyperglycaemia causes is certainly
Dynamic oxidation, the enhancing of protein glycosylation and Anomalous lipid metablism are diabetic's interior free yl, particularly active oxygen
(ROS) increased main cause, while also cause the activity of some antioxidases substantially reduce, so that in diabetic's body
In a certain degree of oxidative stress status.Oxidative stress not only take part in the whole of the generation, development and deterioration of diabetes
Process, but also take part in the important pathological change of diabetic complication -- the atherosclerosis (AS) of various organs.Blood vessel
The important composition composition of wall is VSMC (VSMCs), positioned at media and inner membrance.In blood such as atherosclerosis
In pipe lesion, the abnormality proliferation of VSMC is the main cause of vessel wall thickening.The VSMCs of hyperplasia may further result in
The lesion of coronary endometrium, and then there is atherosclerosis.During the occurrence and development of AS, the propagation of VSMCs and
The balanced degree of apoptosis also can occur corresponding change with the progress of lesion.It is the lesion initial stage of AS, cells of vascular wall increasing number, interior
Film is thickened, tube chamber narrows except relevant with VSMCs hyper-proliferatives, and rate of also being died with tune declines causes cellular accumulation, increasing number close
Cut is closed.The tune of smooth muscle cell die deficiency be also inner film injury artery atherosis early stage progress major reason.It is existing
Suffered for want of medical supplies in technology, functional food can adjust the VSMCs abnormality proliferations of high sugar induced, smooth muscle is thin under regulation sugar environment high
The apoptosis of born of the same parents makes it tend to normal condition, to alleviate diabetic complication, delays or prevent the development of AS.
The content of the invention
The main object of the present invention is to provide under a kind of VSMCs abnormality proliferations that can adjust high sugar induced, sugar environment high
The apoptosis of smooth muscle cell and the small peptide with antioxidation activity.
Still a further object of the present invention is to provide the application of the small peptide.
The purpose of the present invention adopts the following technical scheme that realization.
A kind of small peptide, its amino acid sequence such as SEQ ID NO:Shown in 1.Applicant devises multiple small peptide sequences, by giving birth to
Work bioengineering (Shanghai) limited company provides each small peptide.By substantial amounts of screening operation, it is found that amino acid sequence is
AREGEM(SEQ ID NO:1) small peptide (small peptide hereinafter referred to as of the present invention), not only with preferable inoxidizability, and can
The VSMCs abnormality proliferations of high sugar induced are adjusted, adjusting the apoptosis of smooth muscle cell under sugared environment high, it is tended to normal shape
State.
The present invention also provides the small peptide and is preparing functional food, the antiatherosclerosis shape of alleviation diabetic complication
Into functional food, anti-oxidation function product, the application removed in free base product, anti-lipid peroxidation product.
The present invention is also provided and contains the small peptide, the functional food for alleviating diabetic complication.
The present invention is also provided and contains the small peptide, the functional food formed for antiatherosclerosis.
The present invention also provides the anti-oxidation function product containing the small peptide.
The present invention also provides the free base product of removing containing the small peptide.
The present invention also provides the anti-lipid peroxidation product containing the small peptide.
Beneficial effect：
Small peptide of the present invention, not only with stronger oxidation resistance, and can adjust the VSMCs increasings extremely of high sugar induced
The apoptosis of smooth muscle cell under sugar environment high is grown, adjusted, it is tended to normal condition, therefore, it is possible to alleviate diabetes simultaneously
Hair disease, delays or prevents the development of AS.The present invention also provides the small peptide and is preparing the function food of other complication of alleviation diabetes
Functional food, anti-oxidation function product, the free base product of removing, anti-lipid peroxidation that product, antiatherosclerosis are formed
The application of the aspects such as product.
Brief description of the drawings
Influence of the small peptide of the present invention of Fig. 1 (a) various concentrations to the VSMCs propagation of high sugar induced, wherein G represents sugar high
Group, G+7.5/15/30/45/60 represents the experimental group for adding various concentrations small peptide of the present invention, and numeral is represented and adds the final of small peptide
Concentration；The influence of b VSMCs survival rates that the small peptide of the present invention of () various concentrations is incubated to normal glucose, abscissa is small for the present invention
The ultimate density of peptide.##：There is difference in height (p compared with normal group<0.01)；*：There were significant differences compared with sugar group high (p<
0.05)；**：There is difference in height (p compared with sugar group high<0.01), similarly hereinafter.
The small peptide of the present invention of Fig. 2 various concentrations is to T-AOC in VSMCs (Fig. 2 (a))), GSH-Px (Fig. 2 (b)), SOD (Fig. 2
(c)), the influence of MDA (Fig. 2 (d)) level, wherein G represents sugar group high, and G+7.5/15/45 represents the addition various concentrations present invention
The experimental group of small peptide, numeral represents the final concentration of the small peptide of the present invention for adding.
Influence of the small peptide of the present invention of Fig. 3 (a) various concentrations to ROS in the VSMCs of high sugar induced, wherein G represents sugar high
Group, G+7.5/15/30/45 represents the experimental group for adding various concentrations small peptide of the present invention, and numeral is represented and adds the final dense of small peptide
Degree；The influence of ROS in b VSMCs that the small peptide of the present invention of () various concentrations is incubated to normal glucose, abscissa is small peptide of the present invention
Ultimate density.
Influence of Fig. 4 small peptides of the present invention to Apoptosis, Fig. 4 (a) represents normal group, and Fig. 4 (b) represents sugar group high；Fig. 4
C () represents the experimental group for adding final concentration of 15 μM of small peptides of the present invention；Fig. 4 (d) is represented and is added final concentration of 45 μM of this hairs
The experimental group of bright small peptide；The apoptosis rate of each experimental group cell of Fig. 4 (e) statistical analysis, wherein G represents sugar group high, G+15/45 tables
Show the experimental group for adding various concentrations small peptide of the present invention, numeral represents the ultimate density for adding small peptide of the present invention.
Specific embodiment
1 material：ABTS, fluorescein sodium salt FL, AAPH, Trolox, tetrazolium bromide (MTT), fluorescence probe DCFH-DA, diformazan
Base sulfoxide (DMSO), 45% glucose are purchased from Sigma Co., USA；VSMC (VSMCs) is purchased from Centers For Disease Control And Prevention In Beijing
Center；Hyclone (FBS), low sugar DMEM culture mediums (sugar content is 1g/L), Penicillin-Streptomycin (100
×), Trypsin EDTA (0.05%), Hank balanced salt solutions (HBSS) be purchased from Gibco companies of the U.S.；TAC
(T-AOC) kit, superoxidase (SOD) kit, glutathione peroxidase (GSH-Px) kit, MDA
(MDA) kit, trace of albumin assay (BCA methods) kit build up Bioengineering Research Institute purchased from Nanjing；Annexin
V-FITC cell apoptosis detection kits, RIPA cell pyrolysis liquids are purchased from green skies biological reagent company.
2 key instruments and equipment：Spectra Max M2e multi-function microplate readers, Bio Tek companies of the U.S.；MSC-
Advantage-1.2 Biohazard Safety Equipments, HERA-cell CO2Incubator, Thermo Fisher companies of the U.S.；The miniature whirlpools of WH-2
Whirlpool mixed instrument, Shanghai Hu Xi analytical instrument Co., Ltd., Factory；The Ultrasonic cell smashs of JY92- II, the new skill ultrasonic device in Ningbo
Co., Ltd；THZ-C constant temperature culture oscillators, Taicang experimental facilities factory；H1850R high speed freezing centrifuges, Hunan Hunan instrument reality
Yan Shi instrument developments Co., Ltd；Flow cytometer Accuri C6, U.S. company BD.
The inoxidizability of the small peptide of the present invention of embodiment 1
The present embodiment investigates the inoxidizability of small peptide of the present invention.
1. oxygen radical absorbability (ORAC) is determined
Draw 20 μ L testing sample solutions (ultimate density in reaction system is 50 μM) and 80 μ L Fluresses are (anti-
The ultimate density for answering fluorescein sodium in system is 175nM) in each hole of the microwell plate of black 96, mix, it is incubated 15min at 37 DEG C
Afterwards.100 μ L AAPH (azo-bis-isobutyrate hydrochlorides, in reaction system are added in each hole with from multichannel pipettor to microwell plate
AAPH ultimate densities are 24mM) start reaction.Then microwell plate is positioned in ELIASA, vibration is mixed after 37 DEG C, 485nm
It is measured under excitation wavelength, 538nm launch wavelengths.First order fluorescence intensity is determined per 2min, vibration plate 2s before each reading is determined
After fluorescent value decays to baseline, setting time is 100min to set of time.
It is bent that the Trolox (watermiscible vitamin E) for being respectively 0,6.25,12.5,25,50,100 μM with concentration makes standard
Line.Standard items Trolox and testing sample (ultimate density is 50 μM) dissolved with the phosphate buffer (pH 7.4) of 75mM and
Dilution.Each experimental group setting 6 is parallel, and replaces sample as a control group with phosphate buffer.ORAC values it is general with
Trolox equivalents (Trolox equivalent, TE) represent that unit is μM TE/ μM of Peptides.Result discovery, this hair
Bright small peptide (such as SEQ ID NO:Shown in 1) ORAC values be 2.13 ± 0.07 μM of TE/ μM of Peptides.
2.ABTS+Free radical scavenging activity is determined
Over cure potassium solution (the final concentration of 2.45mM of over cure potassium in storing solution) is prepared with distilled water, it is then molten with this
Liquid obtains ABTS with ABTS (2,2- connection nitrogen-two (3- ethyls-benzothiazole -6- sulfonic acid) di-ammonium salts) aqueous solution mixing, preparation+·
Storing solution (ABTS+Concentration is 7mM).After lucifuge stands 24h at ambient temperature, with the phosphate buffer of 0.1M (PBS,
PH 7.4) by ABTS+Storing solution dilutes certain multiple, makes its absorbance under 734nm 0.7 ± 0.02, obtains
ABTS+Working solution.Experimental group：The ABTS of 190 μ L is drawn with pipettor+In working solution to each hole of 96 orifice plates, then add per hole
Enter the sample solution of 10 μ L, lucifuge reacts 20min in the environment of 25 DEG C after being well mixed, and reaction system is determined after the completion of reaction
Light absorption value under 734nm.Control group：Sample solution is substituted with the phosphate buffer (PBS, pH 7.4) of 0.1M.ABTS+·
The clearance rate of free radical=[(A0-A)/A0] × 100%, wherein, A0Represent the light absorption value of control group；A represents the extinction of experimental group
Value.
The various concentrations of table 1 small peptide of the present invention removes ABTS+Free radical ability (X ± S, n=3) %
Note：X in table 1 represents average, and S represents standard deviation.
As it can be seen from table 1 increase of the small peptide of the present invention with concentration, it removes ABTS+Free radical ability gradually increases
By force, concentration be 5,7.5,10,12.5,15mM when, its clearance rate is respectively 11.70 ± 3.45%, 25.90 ± 4.27%,
36.05 ± 3.26%, 55.65 ± 5.12%, 73.10 ± 4.68%.
Protective effect of the small peptide of the present invention of embodiment 2 to the VSMCs abnormality proliferations of high sugar induced
Tested as follows using small peptide of the present invention.
1. tetrazolium bromide (MTT) method detection cell is bred
Taken the logarithm the VSMCs in growth period, and cell suspension is adjusted into 5 × 10 with the low sugar DMEM culture mediums containing 2%FBS4Individual/
ML, is inoculated in 96 orifice plates, (blank control group adds low sugar DMEM culture mediums of the 100 μ L containing 2%FBS), cell per the μ L of hole 100
Number is 5 × 103Individual/hole, is placed in 37 DEG C, 5%CO2After cultivating 48h in incubator, nutrient solution is sucked, add 100 μ L low per hole
Sugared DMEM culture mediums.Blank control group is not added with any material, and other holes are randomly divided into three big groups：Normal group is without any thing
Matter；Sugar group (G) high adds the glucose solution of appropriate 45%, makes glucose ultimate density be 25mM；Experimental group adds final
Concentration for 25mM glucose and various concentrations small peptide of the present invention, every group set 6 it is parallel.After continuing to cultivate 24h, added per hole
The MTT solution (PBS solution is prepared, and crosses 0.22 μm of sterile film degerming) of 10 μ L, 5mg/mL, cultivates 4h；Abandon supernatant and add 100 μ L
Isopropanol lysate, in 37 DEG C of constant temperature oscillators after lucifuge reaction 15min, light absorption value is determined in ELIASA at wavelength 570nm
(A).Cell proliferation rate=[(As-A0)/(An-A0)] × 100%, wherein, As is experimental group light absorption value, and An is normal group extinction
Value, A0It is blank control group light absorption value.Each data are represented using X ± S (X represents average, and S represents standard deviation) form.
The influence that Fig. 1 (a) breeds for the small peptide of the present invention of various concentrations to the VSMCs of high sugar induced.Using normal group as
Control group, can be obviously promoted the propagation of VSMCs under sugar environmental condition high, cell quantity is higher by the 42.58 ± 3.78% of control group
(p ＜ 0.01).After (7.5,15,30,45 and 60 μM) of the small peptide of the present invention of various concentrations is incubated 24h, cell proliferation rate point
Do not drop to 138.93 ± 5.14%, 132.64 ± 4.76%, 122.55 ± 5.91%, 104.41 ± 4.36% and 103.95 ±
5.29%.When the concentration of small peptide of the present invention is 45,60 μM, the proliferation rate of VSMCs has nearly reached the water consistent with normal group
It is flat.Test result indicate that, certain density small peptide of the present invention can protect cell, effectively suppress the VSMCs propagation that sugar high causes
It is too fast.
In order to understand fully whether growth of the small peptide of the present invention on VSMCs has influence, have detected under normal glucose incubation conditions
(normal group i.e. in the present embodiment title 1 in first paragraph) adds influence of the various concentrations small peptide of the present invention to VSMCs survival rates,
Shown in result such as Fig. 1 (b).From this figure, it can be seen that under 7.5,15,30,45 and 60 μM of concentration conditions, small peptide pair of the present invention
The growth of the VSMCs that normal glucose is incubated illustrates that 7.5-60 μM of small peptide of the present invention is not any to cell without any harmful effect
Toxicity (p>0.05), to VSMCs both without promoting growth, also unrestraint growth.
2. cell TAC (T-AOC), anti-oxidant enzyme activity (glutathione peroxidase GSH-Px and super oxygen
Thing mutase SOD) and MDA (MDA) detection
Take the logarithm the VSMCs in growth period, after being digested with 0.05%EDTA- trypsin solutions, with the low sugar containing 10%FBS
Cell suspension is adjusted to 1 × 10 by DMEM culture mediums5Individual/mL, is inoculated in 24 orifice plates, per the μ L of hole 500, makes cell number be 5 × 104/
Hole, after cell is completely adherent, uses the low sugar DMEM medium culture 24h without serum instead, makes cells Synchronous in G0Phase.At random
It is divided into three big groups：Normal group is without any material；Sugar group (G) high adds the glucose solution of appropriate 45%, makes glucose
Ultimate density be 25mM；It is the glucose of 25mM and the small peptide of the present invention of various concentrations, every group that experimental group adds ultimate density
If 6 parallel.Continue to be incubated 48h.Then supernatant is removed, each hole adds 200 μ L cell pyrolysis liquids, on ice cell lysis
After 15min, scraped with cell and scrape off cell and lysate, be transferred in centrifuge tube, then the ultrasonic wave under the conditions of ice-water bath
Broken (power 300W, 3~5s/ times, is spaced 10s, is repeated 3 times), draws each group cell homogenates liquid, by kit (total antioxidation
Ability (T-AOC) kit, superoxidase (SOD) kit, glutathione peroxidase (GSH-Px) kit, the third two
Aldehyde (MDA) kit is purchased from Nanjing and builds up Bioengineering Research Institute) specification determines the T- of each experimental group cell pyrolysis liquid
AOC, SOD and GSH-Px activity and MDA contents, each number is represented using X ± S (X represents average, and S represents standard deviation) form
According to.
Fig. 2 (a) is influence of the small peptide of the present invention of various concentrations to T-AOC contents in VSMCs.TAC (T-
AOC it is an index that can comprehensively reflect oxidation resistance) including the whole polyphenoils including enzyme and non-enzymatic.By
After the glucose treatment of 25mM, the T-AOC in VSMCs is significantly reduced (p compared with normal group<0.01).The sheet of low concentration
Invention small peptide has the effect of the T-AOC that improves, but DeGrain (p>0.05).Compared with sugar group high, concentration is added
After 15 μM and 45 μM of small peptides of the present invention, the T-AOC in VSMCs be respectively increased 6.31 ± 0.34U/mg and 7.25 ±
0.39U/mg(p<0.01).Show that small peptide of the present invention can protect cell by improving cellular anti-oxidant capacity.
From Fig. 2 (b) and 2 (c), GSH-Px and the SOD activity in normal group VSMCs are passed through in level higher
After the glucose treatment 48h of 25mM, GSH-Px the and SOD activity in sugar group (G) cell high significantly reduces (p<0.01)).With high
Sugared group is compared, and with the rising of small peptide of the present invention, enzymatic activity has not the activity of GSH-Px and SOD in each experimental group cell
With the raising of degree.The vigor of small peptide group of the present invention (7.5,15 and 45 μM) glutathione peroxidase of variant concentration point
Wei not 119.96 ± 4.30U/mg, 129.47 ± 4.09U/mg, 141.04 ± 4.23U/mg；The vigor of superoxide dismutase point
Wei not 127.87 ± 5.16U/mg, 142.29 ± 4.88U/mg, 159.79 ± 5.54U/mg.Statistic analysis result shows, 15 μM
There is pole conspicuousness to improve (p with the enzymatic activity of 45 μM of GSH-Px and SOD enzyme activity higher sugar groups of small peptide experimental group of the present invention<
0.01)。
MDA (MDA), the lipid peroxidation that the polyunsaturated fatty acid attacked in biomembrane as oxygen radical triggers
The final product of reaction, the change of its content can indirectly reflect the extent of damage of cell.From Fig. 2 (d).Sugar group high
MDA contents significantly raise (p compared with normal group<0.01)), illustrate that sugar environment high makes plasma membrane lipid peroxidating and accumulates big volume production
Thing MDA.After (7.5,15 and 45 μM) of small peptide of the present invention for adding various concentrations is incubated 48h, the content of the intracellular MDA of each group
1.40 ± 0.05,1.17 ± 0.04 and 0.87 ± 0.04nmol/mg are dropped to respectively.Statistic analysis result shows, various concentrations
(7.5,15 and 45 μM) small peptide of the present invention to the MDA contents in cell have extremely significant scavenging action (p<0.01).Cell
The change of interior MDA contents, shows that small peptide of the present invention inhibits intracellular lipid peroxidation to a certain extent, enhances
The Antioxidative Defense System of cell.
The detection of active oxygen (ROS) content in 3.VSMCs
Take the logarithm the VSMCs in growth period, after being digested with 0.05%EDTA- trypsin solutions, with the low sugar containing 10%FBS
Cell suspension is adjusted to 1 × 10 by DMEM culture mediums5Individual/mL, is inoculated in 24 orifice plates, per the μ L of hole 500, makes cell number be 5 × 104/
Hole, is placed in 37 DEG C, 5%CO2After cultivating 48h in incubator, nutrient solution is sucked, use the low sugar DMEM culture mediums without serum instead.
After 16h, three big groups are randomly divided into：Normal group is without any material；Sugar group (G) high adds the glucose of appropriate 45% molten
Liquid, makes glucose ultimate density be 25mM；It is the glucose of 25mM and the present invention of various concentrations that experimental group adds ultimate density
Small peptide, every group set 3 it is parallel.After continuing to cultivate 8h, liquid in hole is abandoned in suction, and 2 are cleaned with HBSS (Hank balanced salt solutions) solution
After, the fluorescence probe DCFH-DA (2', 7'- dichlorofluorescein diacetate) for adding 300 μ L concentration to be 10 μM per hole is molten
Liquid, is put into CO2After 1h being cultivated in incubator；Fluorescence is determined at excitation wavelength 485nm, launch wavelength 530nm in ELIASA
Value.Each data are represented using X ± S (X represents average, and S represents standard deviation) form.
Shown in experimental result such as Fig. 3 (a).Content with the intracellular ROS of normal group is 100%, high as control group
The intracellular ROS contents of sugar group are significantly higher than normal group (p<0.01), its intracellular ROS content reached 118.32 ±
2.85%.By after (7.5,15,30 and 45 μM) treatment of small peptide of the present invention of various concentrations, the intracellular ROS levels of each group are divided
105.24 ± 3.32%, 98.28 ± 4.63%, 96.39 ± 3.59% and 96.96 ± 4.23% are not dropped to.Experimental result table
It is bright：By the way that after the small peptide of the present invention for adding various concentrations in sugar high, compared with sugar group high, intracellular ROS can be significantly reduced
Level.Illustrate that small peptide of the present invention can effectively suppress ROS levels in the VSMCs that sugar high causes and increase, suppress the hair of oxidative stress
It is raw.
In order to probe into small peptide of the present invention in itself to the influence of ROS contents in VSMCs, we have detected and are incubated bar in normal glucose
(normal group of first paragraph in the present embodiment title 3) adds the content of ROS in VSMCs after various concentrations small peptide of the present invention under part.
From Fig. 3 (b) as can be seen that adding the experimental group and the intracellular ROS contents of normal group of various concentrations small peptide of the present invention without notable
Sex differernce (p>0.05), it was demonstrated that the content of ROS is not any in the VSMCs that certain density small peptide of the present invention is incubated to normal glucose
Influence.Test result indicate that small peptide of the present invention is in itself to the noiseless effect of experimental result.Prove simultaneously, small peptide of the present invention is to sugar high
The protective effect of the VSMCs of induction is realized by suppressing oxidative stress.
Small peptide of the present invention can not only significantly improve TAC (T-AOC), gluathione in the VSMCs that sugar high is incubated
The activity of peptide peroxidase (GSH-Px) and superoxide dismutase (SOD), reduces lipid peroxidation product MDA (MDA)
Content, effectively suppress active oxygen (ROS) level in the VSMCs that sugar high causes and increase, suppress oxidative stress generation, Er Qieneng
Enough suppress the vascular smooth muscle cell proliferation of high sugar induced.
The influence of the VSMCs apoptosis that the small peptide of the present invention of embodiment 3 is incubated to sugar high
Tested as follows using small peptide of the present invention.
The double dye method detection apoptosis rates of Annexin V-FITC/PI：The apoptosis rate of each group cell uses Annexin V-
FITC cell apoptosis detection kits (green skies biological reagent company), the i.e. double dye methods of Annexin V-FITC/PI.Take the logarithm life
Long-term VSMCs, after being digested with 0.05%EDTA- trypsin solutions, with the low sugar DMEM culture mediums containing 10%FBS by cell
Suspension is adjusted to 5 × 104Individual/mL, is inoculated in 6 orifice plates, per hole 2mL, makes cell number be 1 × 105Individual/hole, be placed in 37 DEG C, 5%
CO2Incubator in cultivate 48h after, suck nutrient solution, use the low sugar DMEM culture mediums without serum instead, and it is big to be randomly divided into three
Group：Normal group is without any material；Sugar group (G) high adds the glucose solution of appropriate 45%, makes the final dense of glucose
It is 25mM to spend；It is the glucose of 25mM and the small peptide of the present invention of various concentrations that experimental group adds ultimate density, and every group sets 3 and put down
OK.After continuing to cultivate 48h, cell culture fluid is suctioned out into centrifuge tube, remove supernatant with PBS washed cell once
After liquid, digested with 0.05%EDTA- trypsin solutions and collect cell, add the cell of collection to train after absorbing trypsin solution
Nutrient solution, slightly mixes, and is transferred in centrifuge tube, by cell suspension at 4 DEG C, with 800g centrifugations 5min after abandon supernatant, collect cell, so
Afterwards with PBS gently re-suspended cell, 5min is centrifuged at 4 DEG C, with 800g, removes supernatant, add 195 μ L Annexin
V-FITC combinations liquid gently re-suspended cell, is subsequently adding 5 μ L Annexin V-FITC and 10 μ L propidium iodide stain liquid, gently
Mix, lucifuge is incubated 20min at room temperature.It is subsequently placed in ice bath, flow cytomery cell, the number of acquisition is carried out immediately
It is X-axis according to Annexin V-FITC fluorescence intensities (FLl-A passages), PI is that fluorescence intensity (FL2-A passages) is the scatterplot of Y-axis
Map analysis Apoptosis situation.
Fig. 4 is influence of the small peptide of the present invention to Apoptosis.The each group cell overwhelming majority appears in Annexin-/PI -It is double
Negative areas, illustrate that cell is in normal condition.Compared with normal group cell, sugar group Annexin high+/PI -Cell and
Annexin+/PI+Cell proportion declines, and illustrates that sugar environment high suppresses the apoptosis of smooth muscle cell.Add the present invention of various concentrations
After small peptide treatment, Annexin+/PI-Cell and Annexin+/PI+The ratio of cell is gradually increasing.Test result indicate that：This hair
Bright small peptide can weaken this inhibitory action.Further analysis each group cell apoptosis rate (each data are represented using X ± S-shaped formulas,
X represents average, and S represents standard deviation).Add 25mM glucose treatment after, apoptosis rate by normal group 21.30 ±
1.41% 14.20 ± 0.88% (p for being reduced to sugar group high<0.01) small peptide of the present invention (15 (M and 45 (M) incubations 48h, are added
Afterwards, apoptosis rate rises to 17.60 ± 0.85% (p respectively<0.05), 20.80 ± 1.13% (p<0.01).Experimental result table
It is bright：Sugar environment high suppresses the apoptosis of smooth muscle cell, and small peptide of the present invention can weaken this inhibitory action.
In sum, small peptide of the present invention has good antioxidation activity, and the VSMCs that can adjust high sugar induced increases extremely
Growing makes it be intended to normal condition, and the apoptosis that can adjust smooth muscle cell under sugar environment high tends to normal condition, therefore, this hair
Bright small peptide can apply to prepare alleviate the functional food of diabetic complication, antiatherosclerosis and formed functional food,
In anti-oxidation function product, the free base product of removing and anti-lipid peroxidation product.
SEQUENCE LISTING
<110>Nanjing University of Finance and Economics
<120>A kind of small peptide and its application
<130> 20161206
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 6
<212> PRT
<213> artificial
<220>
<223>Small peptide
<400> 1
Ala Arg Glu Gly Glu Met
1 5
Claims (7)
1. a kind of small peptide, its amino acid sequence such as SEQ ID NO:Shown in 1.
2. small peptide described in claim 1 is preparing what the functional food of alleviation diabetic complication, antiatherosclerosis were formed
Functional food, anti-oxidation function product, the application removed in free base product, anti-lipid peroxidation product.
3. small peptide, the functional food for alleviating diabetic complication described in claim 1 are contained.
4. small peptide, the functional food formed for antiatherosclerosis described in claim 1 are contained.
5. the anti-oxidation function product containing small peptide described in claim 1.
6. the free base product of removing containing small peptide described in claim 1.
7. the anti-lipid peroxidation product containing small peptide described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611116452.1A CN106749525B (en) | 2016-12-07 | 2016-12-07 | Small peptide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108299550A (en) * | 2018-03-07 | 2018-07-20 | 南京财经大学 | A kind of small peptide and its application with antioxidant activity |
| CN111499707A (en) * | 2020-04-20 | 2020-08-07 | 南京财经大学 | Peptide for reducing blood sugar and blood fat and application thereof |
| CN112175057A (en) * | 2020-09-30 | 2021-01-05 | 南京财经大学 | Antioxidant peptide with liver protection effect |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012098546A2 (en) * | 2011-01-20 | 2012-07-26 | Oneday - Biotech And Pharma Ltd. | Antioxidant, anti-inflammatory, anti-radiation, metal chelating compounds and uses thereof |
| CN102898504A (en) * | 2012-10-23 | 2013-01-30 | 中国农业大学 | Antioxidation active synthetic peptide and purpose thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012098546A2 (en) * | 2011-01-20 | 2012-07-26 | Oneday - Biotech And Pharma Ltd. | Antioxidant, anti-inflammatory, anti-radiation, metal chelating compounds and uses thereof |
| CN102898504A (en) * | 2012-10-23 | 2013-01-30 | 中国农业大学 | Antioxidation active synthetic peptide and purpose thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108299550A (en) * | 2018-03-07 | 2018-07-20 | 南京财经大学 | A kind of small peptide and its application with antioxidant activity |
| CN111499707A (en) * | 2020-04-20 | 2020-08-07 | 南京财经大学 | Peptide for reducing blood sugar and blood fat and application thereof |
| CN112175057A (en) * | 2020-09-30 | 2021-01-05 | 南京财经大学 | Antioxidant peptide with liver protection effect |
| CN112175057B (en) * | 2020-09-30 | 2022-04-29 | 南京财经大学 | Antioxidant peptide with liver protection effect |
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