CN108159037A - A kind of unsaturated fatty-acid compositions and its application for being used to improve neuroprotective function - Google Patents
A kind of unsaturated fatty-acid compositions and its application for being used to improve neuroprotective function Download PDFInfo
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- CN108159037A CN108159037A CN201711468538.5A CN201711468538A CN108159037A CN 108159037 A CN108159037 A CN 108159037A CN 201711468538 A CN201711468538 A CN 201711468538A CN 108159037 A CN108159037 A CN 108159037A
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- unsaturated fatty
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
- A23L33/12—Fatty acids or derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The present invention relates to a kind of for improving the unsaturated fatty-acid compositions of neuroprotective function, it is characterised in that：EPA:The molar ratio of DHA is 1:2.The invention further relates to a kind of for improving the health food of neuroprotective function, it is characterised in that：Containing unsaturated fatty-acid compositions described in claim 1, in the unsaturated fatty-acid compositions, EPA:The molar ratio of DHA is 1:2.The present invention combines by adjusting ratio EPA and DHA, the Neuroprotective effect being optimal, and maximally efficient ratio combination is therefrom determined.And ratio formula can be optimal by manually reconciling, so as to provide product function effect.
Description
Technical field
The present invention relates to a kind of for improving unsaturated fatty-acid compositions and its application of neuroprotective function, and then,
It is related to health food, food nutrition, drug field.
Background technology
Epidemiological survey has proven to the low incidence of AD (senile dementia) and the diet ω -3PUFAs of intake high-content,
Mainly eicosapentaenoic acid (EPA) is related to docosahexaenoic acid (DHA).ω -3 long-chain polyunsaturated fatty acids (ω -
It is essential nutriment 3PUFAs) as the component part of neuron membrane, main make is risen to human organ and its function
With.They participate in inflammation and immunological process and hormone control.In addition, they participate in development and the function of brain.
Numerous studies have proven to oxidative stress and can result in damage and the dysfunction of neuron with long-term chronic inflammation, are
Depression or the inducement of AD morbidities.
Several evidences show that the presence of inflammatory cytokine in AD increases, such as TNF-α level increases in AD patients serums
The presence of (Khemka etc., 2014) and interleukin-6 mRNA in APPsw transgenic mice Tg2576 hippocampus and cortex with
And around the increase of the microglia cell of the activation of senile plaque in cerebral cortex.
The microglia cell of activation can not only generate proinflammatory cytokine, but also can generate free radicals an oxidation
Nitrogen and superoxide anion.These groups induce the neurodegeneration event similar to AD together with TNF-α secretion object.Research shows that
A β in SH-SY5Y cells25-35Toxicity and ROS and NO releases and the enhancing of oxidative damage it is related, it is quick to have raised redox
The transcription factor of sense such as NF- κ B, this is oxidation and an important factor for inflammatory reaction in AD.
In previous studies, it was recently reported that neuroinflamation can reduce the level of neurotrophic factor such as NGF and BDNF.Thing
In reality, it is reported in the concentration of neurotrophic factor and function of receptors increased and decreased in AD patient.Neurotrophy system participates in
With neure growth, survival and the relevant many physiology courses of plasticity.It also reported cholinergic neuron nerve to occur in AD
Dysfunction.
ω -3PUFAs are related to the structure and function that cell membrane phospholipid is formed in brain, and are considered the lifting in cognitive process
It acts on.Secondly, it has been found that ω -3PUFAs have anti-oxidant and anti-inflammatory effect.Particularly in the brain of aging, this spy
Property potentially contributes to the protection of neuron and prevents cell death.
At present, the fish oil comprising unrighted acid or linseed oil have been widely used in health food.However facing
In bed and experiment, the effect of different unrighted acids is not consistent：There is good anti-inflammatory, the anti-oxidant, neuroprotection of display,
There is invalid report.These different effects may it is related with its separate sources insatiable hunger aliphatic acid used or with its source difference institute
The ratio difference containing DHA with EPA leads to the difference of result.
Invention content
The purpose of the present invention is to provide a kind of for improving the unsaturated fatty-acid compositions of neuroprotective function, comprising
EPA and DHA, it is characterised in that：EPA:The molar ratio of DHA is 1:2.
It is an object of the invention to also provide a kind of health food for being used to improve neuroprotective function, it is characterised in that：
Containing unsaturated fatty-acid compositions of the present invention, in the unsaturated fatty-acid compositions, EPA:The molar ratio of DHA is
1:2。
The present invention further provides the unsaturated fatty-acid compositions in the drug for improving neuroprotective function
Using.
The present invention provides application of the unsaturated fatty-acid compositions in the food for improving neuroprotective function.
The present inventor has studied the anti-oxidant, anti-inflammatory and neural of different proportion DHA and EPA ratio combination by systematic comparison
Protecting effect therefrom determines maximally efficient ratio combination.And ratio formula can be optimal by manually reconciling, so as to carry
For product function effect.
Description of the drawings
Fig. 1 show to prepare for improve neuroprotective function unsaturated fatty-acid compositions flow chart.
The influence that cell viability EPA, DHA of Fig. 2 a various concentrations beta induced to A reduces.
The influence that Fig. 2 b EPA, DHA and its different proportion cell viability beta induced to A reduce.
The protective effect of Fig. 2 c EPA, DHA and its different proportion cellular damage beta induced to A.
The influence of Fig. 3 a EPA, DHA and its different proportion the ROS level variation beta induced to A.
The influence of Fig. 3 b EPA, DHA and its different proportion the NO level variation beta induced to A.
The influence of Fig. 3 c EPA, DHA and its different proportion the GSH level variation beta induced to A.
Fig. 4 a EPA, DHA and its different proportion the TNF-α mRNA beta induced on A express increased influence.
The horizontal increased influence of Fig. 4 b EPA, DHA and its different proportion TNF-α beta induced on A.
The influence of Fig. 5 a EPA, DHA and its different proportion the NGF mRNA expression beta induced to A.
The influence of Fig. 5 b EPA, DHA and its different proportion the BDNF mRNA expression beta induced to A.
The influence of Fig. 5 c EPA, DHA and its different proportion the NGF level beta induced to A.
The influence of Fig. 5 d EPA, DHA and its different proportion the BDNF level beta induced to A.
Fig. 6 a EPA, DHA and its different proportion Bax beta induced to A：The influence of Bcl-2 ratios.
The influence of Fig. 6 b EPA, DHA and its different proportion the Caspase-3 protein expression beta induced to A.
Specific embodiment
Below in conjunction with the specific embodiment of the invention, technical scheme of the present invention is verified, the embodiment verified
Only section Example of the invention.Based on the embodiments of the present invention, this field researcher is not making any creation
Property labour under the premise of the other embodiment that is obtained, shall fall within the protection scope of the present invention.
In the unsaturated fatty-acid compositions for being used to improve neuroprotective function of the present invention, EPA:The molar ratio of DHA is
1:2。
EPA, DHA wherein in composition are commercially available sterling, and the preparation flow of composition is as shown in Figure 1：It surveys respectively first
The influence of the cell viability of the AD cell models of EPA and DHA the SH-SY5Y cell beta induced to A of various concentration is tried, so as to really
Surely the dose concentration to shield, EPA and DHA processing cells then again in varing proportions, detects the thin of SH-SY5Y cells
Born of the same parents' vigor, Antioxidant Indexes：ROS, NO, GHA, anti-inflammatory index：TNF-α, anti-apoptotic index Bc1, Bax, Caspase-3, so as to
Determine EPA：The molar ratio of DHA is 1:Neuroprotective effect is optimal when 2, only need to manually allocate EPA and DHA to required ratio,
It is uniformly mixed using conventional method and obtains the composition.
It is appreciated that the unsaturated fatty-acid compositions of the present invention, can individually eat as health food, can also add
Enter into other food mixed edible or applied in the drug for improving neuroprotective function is prepared.
The health food for being used to improve neuroprotective function of the present invention, contains the insatiable hunger described in the first aspect of the present invention
And aliphatic acid composition, in the unsaturated fatty-acid compositions, EPA:The molar ratio of DHA is 1:2.
The health food can be the form of ordinary food, can also use tablet, capsule, pill, lyophilized form or
Any suitable administration form of person.
Health food in the scope of the invention may include the additive below one or more：Preservative, solubilizer, stabilization
Agent, wetting agent, emulsifier, sweetener, colorant, flavoring agent, deodorant, buffer solution, coating agent, antioxidant, suspending agent,
Auxiliary agent, excipient and diluent.
The present invention further provides the unsaturated fatty-acid compositions in the drug for improving neuroprotective function
Using.
The unsaturated fatty-acid compositions containing EPA and DHA can be used in pharmaceutical composition so that rich in drug
Containing EPA and DHA.
The convenient form of the pharmaceutical composition can be tablet for oral administration, pill, capsule, syrup
Agent, pulvis or granule；Sterile parenteral or subcutaneous solution for parenterai administration, suspension or the bolt for rectally
Agent, all these is all well known in the art.
Specific dosage level and dose frequency can be variation for any specific patient, will depend on more
Kind of factor, the metabolic stability of activity, this compound activity including used specific compound and duration, the age,
Weight, general health, gender, diet, the mode of administration and time, the rate of excretion, the combining of drug, specific disease
The severity of disease and the treatment of single progress.The medicament and/or pharmaceutical composition of the present invention can be according to daily 1~10 time
Administration, such as once or twice daily.For oral and parenteral administration patient, the dosage level of medicament can be single dose
Amount or separated dosage.
The present invention provides application of the unsaturated fatty-acid compositions in the food for improving neuroprotective function.
The composition of the present invention can be used in food industry so that food (such as grain food, dairy products, soya-bean oil, beans
Slurry) in be rich in EPA and DHA.
The cereal foods can have different shape or appearance forrns.For example, stick, cake can be made into
Or macaroon or its can also be oatmeal shape, sheet or rodlike.It can individually eat or with dairy products such as milk, acid
Milk or cottage cheese etc. are eaten together.
It should be understood that essence can also be added in food, such as honey, fruit, chocolate, caramel, nut, apricot
Benevolence, yoghurt flavours or combination.
Current market sales of unsaturated fat acid product also has largely from deep sea fish oil from plant, various
The extraction of algae, ingredient is different, and especially there are notable differences in DHA and EPA ratios.It is such as common from gold in the market
Fish oil DHA in prosperous fish or dog salmon：EPA is close to 1:2；And the unrighted acid in seaweed then contains higher DHA ratios
Example, this leads to health effect, and there are bigger differences.And so far, it there is no to DHA and EPA ratios group in unrighted acid
It closes in anti-oxidant, anti-inflammatory, the research in terms of neuroprotection, it is unclear between the variation of the two ratio and effect, therefore also without most
Ratio of greater inequality example formula and product.
The present inventor has studied the anti-oxidant, anti-inflammatory and neural of different proportion DHA and EPA ratio combination by systematic comparison
Protecting effect therefrom determines maximally efficient ratio combination.And ratio formula can be optimal by manually reconciling, so as to really
Protect product function effect.Be it in health food or drug with providing reference.
A β be by amyloid precusor protein (APP) through β-and the protein hydrolysate of gamma-secretase under pathological conditions.A
β25-35It is a kind of synthetic peptide for the overall length A β for corresponding to 25-35 amino acid, there is identical beta sheet structure, and keep overall length
Aβ1-42Complete toxicity.The peptide shows to form the rapid aggregation property of stable fibrinogen, and has god immediately in dissolving
Through toxicity.The present invention uses A β25-35The SH-SY5Y cells of differentiation are damaged as AD models, more different EPA/DHA ratios pair
A β in SH-SY5Y cells25-35The potential neuroprotection of the neurotoxicity of induction.
At present, without any experiment respectively to EPA, DHA or a certain proportion of combinations are studied and in same experiment
More different ω -3PUFAs.The present invention studies the combination of individual EPA and DHA and its different proportion to A β25-35The AD of induction
The effect of cell model.Cell viability is measured, to compare EPA, the combination of DHA or its various ratio is to A β25-35The AD of induction
The influence of the neurotoxicity of cell model and their potential synergistic effect.In addition, measure oxidative stress, proinflammatory cytokines because
The level of son and neurotrophic factor, to analyze EPA, DHA or combinations may be beneficial to the cell and molecular mechanism of AD.And
The EPA/DHA of different proportion adjusts the ability of the expression of apoptosis-associated genes.
In the present invention, " FAs " refers to the EPA of various concentration, DHA or the combination of its different proportion.
Embodiment
Human neuroblastoma cell system SH-SY5Y cells all-trans retinoic acid (RA) is handled 7-8 days and is divided completely
Turn to human neure like cell.In the last day of differentiation, cell starts to test.In A β25-35The SH- of the differentiation of induction
EPA and DHA is tested in the AD cell models of SY5Y cells in 6,12,25,50,100 μM of influences to cell viability.Wherein occur
Slight but significant attenuation A β25-35The optimal dose of point selected as EPA and DHA that the cell viability of induction reduces and culture continue
Time.Then carry out following seven groups of researchs：(i) (culture medium) is compareed, (ii) A β25-35(add A β25-35Culture medium), (iii) A β
+ EPA (is pre-processed, then with A β with EPA25-35Processing), (iv) A β+DHA (are pre-processed, then with A β with DHA25-35Processing) and
(v-vii) A β+EPA+DHA are (respectively with 2:1,1:1 and 1:2 EPA+DHA (totally 25 μM) processing, then with A β25-35Processing).
Add A β25-35Before, cell EPA, DHA, combination thereof or control solvent are pre-processed 12 hours.Add in A β25-35(eventually
A concentration of 20 μM) after, cell is incubated 24 hours again.Then, the cell viability in SH-SY5Y cells, oxidative stress, inflammatory are studied
Cytokine TNF-α, neurotrophic factor and Apoptosis.
In the present embodiment, it as reagent, uses>Eicosapentaenoic acid (the EPA of 99% purity；20：5, n-3) and 20
Two carbon acid (DHA；22：6, n-3) sodium salt comes from Sigma-Aldrich companies.By FAs dissolvings in the medium, in nitrogen
It is divided into aliquot under air-flow, and is preserved at -80 DEG C until using.
In the present embodiment, SH-SY5Y comes from ATCC (CRL-2266, Lot.61983120).Cell is being contained 10%
Fetal calf serum (FBS,Canada) and 1% Pen .- Strep DMEM/F12 culture mediums (Add and take
Ventilation 75-cm greatly)2It is cultivated in culture bottle.It is dense with end in the DMEM/F12 containing 3%FBS (culture medium is replaced for every 2 days)
RA (Sigma Aldrich, Canada) the processing SH-SY5Y cells spent for 10 μM are divided into complete human neure for 7-8 days
Like cell.
In the present embodiment, using the 3- (4,5- dimethylthiazole -2) -2 for measuring cell proliferation rate and cell viability,
5- diphenyltetrazolium bromides (MTT) measure cell viability.By cell inoculation in 96 orifice plates, 90 are added in into each hole
μ L cell suspending liquids.After experiment process, cell viability is detected with MTT (ATCC) according to the manufacturer's instructions.Use enzyme mark
Instrument (BioTek, USA) measures optical density in 570nm.The absorbance of control group is considered as the 100% of cell viability.
As detected by mtt assay, A β25-35It lives in the cell for the SH-SY5Y cells that 20 μM significantly reduce differentiation in 24 hours
Power (p<0.01, Fig. 2 a).However, significantly reduce A β with (6-100 μM) pretreatment of EPA or DHA of various concentration25-35With agent
The reduction of cell viability caused by measuring dependence mode, and 6 in EPA, 12 (p>0.05), 25 (p<0.05), 50 μM of (p<
And 100 μM of (p 0.01)<0.05) 6 (p or in DHA<0.05), 12-50 (p<And 100 μM of (p 0.01)<0.05) (Fig. 2 a).
Compared with EPA, the effect of DHA seems more stronger than effects of the EPA under same dose.On the basis of these results, subsequent
In the measure of SH-SY5Y cells progress oxidative stress, inflammation and apoptosis, we select 25 μM as EPA in different proportion processing
With the accumulated dose of DHA combinations.
In the present embodiment, it since MTT is measured to cell quantity sensitivity, is influenced by cell Proliferation and cell viability,
It you must use another detection method and confirm result.Come using CytoTox-96 assay kits (Promega, Canada)
Total release of cytoplasm lactic dehydrogenase (LDH) in culture medium is assessed, this is the result of cell integrity damage.The measure is based on
From 2-P- (iodophenyl) -3- (p-nitrophenyl) -5- phenyltetrazoles chloride (INT, tetrazolium salt) to the coupling of formazan product
Enzymatic conversion, enzyme reaction is discharged from cell by LDH and the diaphorase in substrate mixture is measured is catalyzed.Pass through enzyme mark
Instrument reads absorbance at 490nm.Every group of mean light absorbency is normalized to the percentage of control value.
In order to test different EPA/DHA ratios in neuroprotection have influence in various degree it is assumed that test below
It is middle to be combined using following different FAs：EPA/DHA 2：1, EPA/DHA 1：1 or EPA/DHA 1：2.Fig. 2 b's the results show that
With A β25-35Group is compared, in all proportions test, different EPA/DHA ratios significantly (p<0.05) cell viability is improved.Protection
SH-SY5Y cells are from A β25-35The FAs effect of the neurotoxicity of induction is：EPA<2：1EPA/DHA<DHA≤1：1EPA/DHA<
1：2EPA/DHA.
Measure (it is the index of cell death) is discharged by LDH and further demonstrates different proportion EPA/DHA to A β25-35
The protective effect (Fig. 2 c) of the SH-SY5Y cellular damages of induction.With reference to said determination as a result, we can obtain knot for certain
By, EPA, DHA and combinations thereof can to varying degrees effective protection differentiation SH-SY5Y cells from A β25-35Induction
Cellular damage, the EPA of best raising cell viability：The ratio of DHA is 1：2EPA/DHA.
In the present embodiment, SH-SY5Y cells are inoculated in 96 orifice plates, 200 μ L cell suspending liquids are added in into each hole.
After experiment process, according to the manufacturer's instructions, with ROS kits in fluorecyte (Sigma Aldrich) quantization cell
The level of interior ROS.Using fluorescence microplate reader (Reader Synergy HT, BioTek Instruments, the U.S.), with lex=
650/lem=675nm fluorescence intensities.According to the manufacturer's instructions, by Griess reagent systems, (Promega adds and takes
Cell intracellular nitric oxide (NO) yield is measured greatly).Absorbance is measured at 540nm using microplate reader.
By SH-SY5Y cell inoculations in 6 orifice plates, and 2mL cell suspending liquids are added in into each hole.In experiment process
Afterwards, according to the manufacturer's instructions, GSH concentration is measured with glutathione assay kit (Sigma Aldrich).Fluorescence intensity
It is measured with fluorimeter reader, excitation wavelength 390nm, launch wavelength 478nm.
Fig. 3 a illustrate A β25-35ROS fluorescence (p is dramatically increased than control group<0.01).When with A β25-35When group compares, individually
(p is significantly reduced using the ROS fluorescence of the combination of EPA and DHA under EPA and all test ratios<0.05).However, in DHA groups
ROS fluorescence does not find significant difference.Protect SH-SY5Y cells from A β25-35The FAs effect for inducing increased ROS fluorescence is：
DHA<1：2EPA/DHA<EPA<1：1EPA/DHA≤2:1EPA/DHA.
As shown in Figure 3b, when individually with A β25-35When handling cell, observe that nitrate levels dramatically increase about 40.29%
(p<0.05).However, with A β25-35Group is compared, and the nitrate levels of EPA processing are used alone slightly but do not significantly reduce (p<
0.05).With A β25-35Group compares, and DHA and the processing of all EPA/DHA processing groups, the equal horizontal significance difference of nitrate-free is used alone
It is different.
It is being shown in Fig. 3 c the result shows that, A β25-35The GSH contents of damaging cells significantly (p<0.05) it reduces, with A β25-35
Group is compared, the EPA/DHA significantly (p of all proportions test<0.05) increase GSH contents.Protect SH-SY5Y cells from A β25-35
Induction anti-oxidant GSH reduce FAs effect be：1：2EPA/DHA≤DHA<1：1EPA/DHA<EPA≤2：1EPA/DHA.
In the present embodiment, by the SH-SY5Y cell inoculations of differentiation in six orifice plates, and addition 2mL is thin into each hole
Born of the same parents' suspension.After experiment, cell is harvested.Follow manufacturer recommendation RNA extraction method (Lipid
Tissue Handbook).Using GoScriptTM Reverse Transcriptase (a) (Promega, Canada) from RNA
Synthesize complementary DNA (cDNA).2 μ gRNA synthesize for the first chain cDNA.The nucleotide sequence of primer is from NCBI's
Nucleotide databases and Primer Premier 6.0.After specificity verification in NCBI- nucleotide-BLAST, draw
The synthesis of object is carried out by Invitrogen companies.It is prepared using Quantitect SYBR Greenmaster mix (Qiagen)
PCR reacts, and carries out PCR reactions using Real Time PCR Detection System (Bio-Rad, the U.S.) CFX96TM real-time systems.PCR processes
It is as follows：95 DEG C are initially incubated 5 minutes to activate Hot-Star-Taq archaeal dna polymerases, then 94 DEG C 15 seconds (denaturation), 59 DEG C 30
Second (annealing) and 72 DEG C 30 seconds (extensions).After 38 cycles, a melting curve is produced, for measuring the spy of primer
The opposite sex and homogeneity.The rna expression (relative quantification) of gene expression dose house-keeping gene beta-actin is corrected with △ △ CT
Standardization.
In the present embodiment, by SH-SY5Y cell inoculations in 6 orifice plates, and 2mL cells is added in into each hole and are suspended
Liquid.After experiment process, cell is collected, and centrifuge 10 minutes, and with RIPA buffer solutions (RIPA, Thermo with 10.000g
Scientific it) cracks.By ultrasonic wave Assisted Cleavage, lysate is centrifuged 10 minutes at 4 DEG C with 10000g.Collect supernatant,
After boiling add in the aliquot containing 20-40 μ g proteins, and on 10%SDS-PAGE gels with 100V in running buffer
It is detached 60 minutes in liquid.Run gel after, by Protein transfer to polyvinylidene fluoride (PVDF) film (Millipore,
Bellerica, MA, the U.S.) on.Then trace is washed 5 minutes in Tris buffer solutions-polysorbas20 (TBST), then 20
(TBST and 5% alipoidic milk power) is closed at DEG C 1 hour.It after closing, washs trace 5 minutes with TBST, is incubated with primary antibody, wrap
It includes for more grams of the rabbit source of actin, NGF, BDNF, TrkA, TrkB, TNF-α, Bcl-2, Bax and Caspase-3 (Abcam)
Grand antibody, 4 DEG C overnight, and secondary antibody, the anti-rabbit IgG of peroxidase (HRP) combination 1 hour are then added at 20 DEG C.By trace
It is washed three times in TBS.Use ClarityTMWestern ECL substrate reagent box (Bio-rad, Canada) are with Image
LabTMThe ChemiDoc of software (Bio-rad, Canada)TMImmunoreactivity band is detected in MP systems.By being normalized
For the beta-actin detected again on same film, then with the percentage calculation of control group, all target proteins are quantified.
Compared with the control group, after 4 hours are incubated, by giving A β25-35, TNF-α mRNA expression dramatically increase (p<
0.05, Fig. 4 a), but with A β25-35Increase (the P of protein expression can not be found before being incubated 12 hours<0.01).However,
With A β25-35Group is compared, and has individually been restored with the EPA and DHA being used in combination to A β25-35The TNF-α mRNA expression of processing reaction
It significantly reduces.(Fig. 4 a) is in addition, 1：The effect of 1EPA/DHA is with obvious effects more more effective than individual EPA or DHA, these evidences
Show 1:1EPA/DHA can play anti-inflammatory agent potential synergistic effect.
It is similar to TNF-α gene, A β25-35Processing occurs apparent ngf gene expression variation for 4 hours.Compared with the control group,
Aβ25-35Damaging cells NGF mRNA expression is apparent to lower (p<0.01, Fig. 5 a).Meanwhile A β25-35Damaging cells BDNF genes
(p was also lowered in mRNA expression at 4 hours<0.01, Fig. 5 b).Compared with the control, A β25-35In damaging cells, when being incubated within 12 hours
It was found that the protein expression of NGF significantly reduces (p<0.01, Fig. 5 c), and bdnf protein expression dramatically increases (p<0.01, Fig. 5 d).With
EPA, DHA and its pretreatment of various ratios can weaken A β to some extent25-35NGF mRNA and the protein expression (figure of induction
5a, c), BDNF mRNA expression (Fig. 5 b) and bdnf protein expression variation (Fig. 5 d).
Different from the gene of front, TrkA and TrkB are giving A β25-35It is affected after 24 hours.Aβ25-35It is substantially reduced
TrkA protein expressions (p<0.01).EPA, DHA and its processing of various ratios cannot significantly change A β25-35Effect to receptor, is removed
2：1EPA/DHA significantly increases this variation (Fig. 5 a).Individually giving A β25-35Cell in, it has been found that TrkB protein
Distant increase (p<0.05).2：1 and 1：2EPA/DHA pretreatments can part significantly reverse A β25-35The TrkB expression of induction
Change (equal p<0.05, Fig. 5 b), compared with A β groups, other groups handled with EPA and/or DHA are without significant change.
In A β25-35Different incubation times (4,8,12 and 24 hours) under test cdna Bax, Bcl-2 and Caspase-3 egg
White expression.Bcl-2 is with A β25-35When being incubated 24 hours, protein expression is by A β25-35It is strong to reduce, by using EPA, DHA and its
The pretreatment of various ratios dramatically increases (p in various degree<0.01).About Bax, do not found after being incubated at 4~24 hours apparent
Variation.Bax in cell：The ratio of Bcl-2 is individually giving A β25-35Dramatically increase (p<0.01).However, EPA, DHA and its
The processing of various ratios can differently be obviously reduced this effect, and by Bax：Bcl-2 ratios are reduced to different degrees of control
Level is (in addition to EPA p<0.05, all p<0.01, Fig. 6 a)
For caspase-3 mRNA, A β25-35Dramatically increase its protein expression (p<0.01), EPA, DHA and its various ratios
Example processing can weaken A β to some extent25-35Effect (in addition to 1：1EPA/DHAp<0.05, all p<0.01, Fig. 6 b).
To sum up, 1：2EPA/DHA shows most effective to cell viability reduction；2：1EPA/DHA is sent out in test group
Wave the most effective ratio of antioxidation.For antiphlogistic effects, 1：1EPA/DHA is the optimal proportion in all test ratios；
When being related to Anti-G value, pure DHA is more more effective than the combination of EPA and any other ratio.Based on these as a result, the knot obtained
By for EPA, DHA and its various ratios differently adjust A in SH-SY5Y cells by differently inhibiting inflammation and oxidative stress
β25-35The neurotoxicity of induction adjusts neurotrophic factor level, so as to weaken Neuron Apoptosis.
Claims (4)
1. it is a kind of for improving the unsaturated fatty-acid compositions of neuroprotective function, include EPA and DHA, it is characterised in that：
EPA:The molar ratio of DHA is 1:2.
2. a kind of health food for being used to improve neuroprotective function, it is characterised in that：Contain unsaturation described in claim 1
Aliphatic acid composition, in the unsaturated fatty-acid compositions, EPA:The molar ratio of DHA is 1:2.
3. a kind of application of unsaturated fatty-acid compositions described in claim 1 in the drug for improving neuroprotective function.
4. a kind of application of unsaturated fatty-acid compositions described in claim 1 in the food for improving neuroprotective function.
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