CN105949262B - 3-hydrogenated pinicolic acid cyanide ethyl ester medicine and application thereof - Google Patents
3-hydrogenated pinicolic acid cyanide ethyl ester medicine and application thereof Download PDFInfo
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- CN105949262B CN105949262B CN201610363662.4A CN201610363662A CN105949262B CN 105949262 B CN105949262 B CN 105949262B CN 201610363662 A CN201610363662 A CN 201610363662A CN 105949262 B CN105949262 B CN 105949262B
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- siberian cocklebur
- ethyl ester
- hydrogenations
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- acid cyanogen
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
Abstract
The invention discloses a 3-hydrogenated pinicolic acid cyanide ethyl ester medicine and application thereof. The medicine comprises a compound with the following structure formula which is as shown in the description. The medicine also comprises pharmaceutically acceptable salts, 3-hydrogenated pinicolic acid cyanide ethyl ester as well as derivatives and pharmaceutically acceptable salts thereof, and pharmaceutically acceptable auxiliary materials or carriers. The 3-hydrogenated pinicolic acid cyanide ethyl ester is a PI3K/mTOR dual inhibitor; the invention also relates to application of the 3-hydrogenated pinicolic acid cyanide ethyl ester medicine to promotion of cell autophagy and application of the 3-hydrogenated pinicolic acid cyanide ethyl ester medicine to preparation of medicines for treating tumors such as gastric cancer, liver cancer, lung cancer, prostatic cancer and nasopharynx cancer and the like, neurodegenerative diseases such as presenile dementia and the like, and diabetes. The 3-hydrogenated pinicolic acid cyanide ethyl ester medicin provides a novel clinical medication choice.
Description
Technical field
The present invention relates to the loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations and its application, it is used as PI3K/mTOR double inhibitors
To promoting cell autophagy to produce pharmacological action, 3- can be hydrogenated into loose Siberian cocklebur acid cyanogen second esters medicine is used to prepare treatment stomach cancer etc.
On the medicine of cancer, Alzheimer's class nerve moving back property row lesion or diabetes.
Background technology
The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is the ester type compound synthesized with the raw material of the loose Siberian cocklebur acid B of three hydrogenations, existing skill
The defect of art is the separation purifying technique of the loose Siberian cocklebur acid cyanogen ethyl ester of purifying and 3- hydrogenations of three hydrogenations pine Siberian cocklebur acid B.
The content of the invention
It is an object of the invention to provide a kind of loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations and its application.
In order to solve the above technical problems, the technical solution adopted in the present invention is：A kind of loose Siberian cocklebur acid cyanogen ethyl ester class of 3- hydrogenations
Medicine, the compound containing following structural formula in such medicine：
Such medicine also includes pharmaceutically acceptable salt, and the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations and its derivative can pharmaceutically connect
The salt received, and pharmaceutically acceptable auxiliary material or carrier.
Wherein, the synthetic method of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is：
1) the equimolar loose Siberian cocklebur acid B of 3- hydrogenations, bromoacetonitrile, potassium carbonate are added in reaction bulb, and adds appropriate acetonitrile to make
It is solvent, after the completion of 85 DEG C of stirring reaction 2h, TCL detection reactions, is spin-dried for solvent, adds water and ethyl acetate, separating and extracting
Three times, ethyl acetate is reclaimed, obtain solids；
2) by the solid method loading of solids, 200-300 mesh normal-phase silica gel column chromatography is separated, with petroleum ether：Ethyl acetate=15:
1 (volume ratio) is mobile phase, obtains white solid powder for 3- hydrogenates pine Siberian cocklebur acid cyanogen ethyl ester.Its reaction equation is as follows.
The invention further relates to purposes of the described loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations in cell autophagy is promoted.
The invention further relates to purposes of the described loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations in cell autophagy is promoted, it is special
Levy and be：The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is PI3K/mTOR double inhibitors.
Treatment stomach cancer, liver cancer, lung cancer, preceding is being prepared the invention further relates to the loose Siberian cocklebur acid cyanogen second esters medicine of described 3- hydrogenations
Application in terms of the tumour medicines such as row gland cancer, nasopharyngeal carcinoma.
Treatment nerve retrograde affection medicine is being prepared the invention further relates to the described loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations
The application in object space face.
Treatment Alzheimer's medicine is being prepared the invention further relates to the described loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations
The application in object space face.
The invention further relates to the described loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations in terms for the treatment of diabetes medicament is prepared
Using.
Brief description of the drawings
Fig. 1 is that 3- hydrogenates loose Siberian cocklebur acid cyanogen ethyl ester Structural Identification hydrogen spectrogram.
Fig. 2 be 3- hydrogenate loose Siberian cocklebur acid cyanogen ethyl ester to the Apoptosis of HGC-27 influence (n=3,), wherein A:Control
Group；B：Loose Siberian cocklebur acid cyanogen ethyl ester (10 μM) of 3- hydrogenations；C：Loose Siberian cocklebur acid cyanogen ethyl ester (20 μM) of 3- hydrogenations；D：The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations
(40μM)。
Fig. 3 is influence of the transmission electron microscope loose Siberian cocklebur acid cyanogen ethyl ester of detection 3- hydrogenations to HGC-27 cell autophagies, wherein a：Control
Group (× 6000), b/c：Autophagic vacuole (× 6000), d:Autophagosome submicroscopic structure (× 15000).
Fig. 4 be 3- hydrogenate loose Siberian cocklebur acid cyanogen ethyl ester to the cell autophagy ratio of HGC-27 influence (n=3,), wherein A:
Control group；B：Loose Siberian cocklebur acid cyanogen ethyl ester (10 μM) of 3- hydrogenations；C：Loose Siberian cocklebur acid cyanogen ethyl ester (20 μM) of 3- hydrogenations；D：The loose Siberian cocklebur acid cyanogen of 3- hydrogenations
Ethyl ester (40 μM).
Fig. 5 is the molecular docking result figure that 3- hydrogenates loose Siberian cocklebur acid cyanogen ethyl ester and mTOR.
Specific embodiment
1-5, embodiment and form below in conjunction with the accompanying drawings, the present invention is furture elucidated.These embodiments are interpreted as only using
In illustrating rather than for limiting the scope of the invention.After the content for having read record of the present invention, ability
Field technique personnel can make various changes or modifications to the present invention, and these equivalence changes and modification equally fall into right of the present invention and want
Seek book limited range.
Experiment material：
1st, cell line used is tested：Human stomach cancer cell line HGC-27, HepG2 cell lines, human lung carcinoma cell line
A549, Breast cancer lines MDA-MB-231, Human carcinoma of prostate cell line PC3, human nasopharyngeal carcinoma cell line CNE-2, people's colon
JEG-3 HT-29, normal gastric mucosa cell line GES-1, nerve cell blastoma SH-SY5Y, buy in Wuhan University
State's Type Tissue Collection, cell line is preserved by laboratory passage.
People's HT-29 cell line PTEN deletion forms, the cell is produced to pass through laboratory passage using siRNA gene silencing modes
Preserve cell line.
2nd, agents useful for same and instrument are tested
1) main experiment reagent is：RPMI-1640 culture medium pulvis, U.S.'s Gibco Products；DMEM medium powders
Agent, U.S.'s Gibco Products；Without the new fetal calf serum of mycoplasma, Hangzhou Chinese holly biomaterial Co., Ltd product；3-
(4,5)-dimethylthiazole -2-2,5- diphenyltetrazolium bromide bromide (MTT), Sigma Co., USA's product；Penicillin, streptomysin,
North China drugmaker product；HEPES, U.S.'s Amresco Products；Trypsase, U.S.'s Amresco Products；Diformazan
Base sulfoxide (DMSO), U.S.'s Amresco Products；Cell DNA content detection kit, the triumphant limited public affairs of base biotechnology share
Department's product；Annexin V-FITC/PI cell apoptosis detection kits, Kai Ji Biotechnology Ltd. product；AO, it is triumphant
Base Biotechnology Ltd. product；Trishydroxymethylaminomethane (Tris), Sigma Co., USA's product；Dodecyl
Sodium sulfonate (SDS), Sigma Co., USA's product；(5 ×) SDS-PAGE albumen sample-loading buffers, green skies biotechnology research
Institute's product；The rapid glue kit of PAGE gel, green skies biotechnology research institute product；BCA method protein quantification reagents
Box (reinforced), green skies biotechnology research institute product；Pvdf membrane, green skies biotechnology research institute product；ECL chemistry hairs
Light detection kit, green skies biotechnology research institute product；Fixing solution used by Western and developer solution, Wuhan Google are biological
Science and Technology Ltd.'s product；Total protein of cell extracts total reagent, Wuhan bio tech ltd of Google product；Twee-20, it is military
Chinese Google bio tech ltd's product；P-PI3K-kinase p85 α rabbit antibody (Tyr508, sc-12929),
Santa Cruz Products；P21rabbit antibody (C-19, sc-397), Santa Cruz Products；p-P70S6K
Rabbit antibody (Thr389), U.S.'s Cell Signaling Products；Beclin-1rabbit antibody (D40C5), the U.S.
Cell Signaling Products；Cyclin D1rabbit antibody (92G2), U.S.'s Cell Signaling Products；
LC3A/B rabbit antibody (D3U4C), U.S.'s Cell Signaling Products；P-mTOR rabbit antibody
(Ser4228), U.S. Cell Signaling Products；The antiantibodys of β-Action one, Wuhan doctor obtains biotech firm's product；
Coomassie brilliant blue：Bio tech ltd's product is built up in Nanjing；The goat anti-rabbit igg of horseradish enzyme mark, Beijing Zhong Shan Golden Bridge
Products；Horseradish enzyme marks goat anti-mouse IgG, Beijing Zhong Shan Golden Bridge Products；Film, Kodak's product；Degreasing
Milk powder, YiLi corporation product；Alloxan, Sigma Products；Is produced from Metformin hydrochloride, Beijing JingFeng Pharmaceutical Co., Ltd
Product；Other reagents are the pure rank of the analysis sold market.Healthy male SD rat and kunming mice, are purchased from SanXia University animal
Experimental center.
2) main laboratory apparatus is：96 orifice plates and 6 orifice plates, are purchased from Corning companies of the U.S.；Vertical pressure steam sterilizing
Pot, is purchased from Japanese HIRAYAMA companies；Ultra-pure water instrument, is purchased from Ai Kepu scientific ＆ technical corporation；Olympus KX41 type inverted microscopes,
It is purchased from Thermo companies of the U.S.；3111CO2Incubator, is purchased from match Mo Feishier Thermo companies of the U.S.；Electrophoresis apparatus, is purchased from Beijing
Biological instrument company of city 61；Stat Fax-2100 type enzyme-linked immunosorbent assay instruments, are purchased from Awareness companies of the U.S.；Ultra-clean work
Make platform, be purchased from Suzhou Decontamination Equipment Plant；Electronic balance, is purchased from Shanghai Min Qiao precision instruments Co., Ltd；5415D mini desktops are high
Fast centrifuge, is purchased from German Eppendof companies；Micropipettor, is purchased from German Eppendof companies；- 80 DEG C of ultralow temperature ice
Case, is purchased from Qingdao Haire Special Electrical Appliances Co., Ltd；Decolorization swinging table STS-1 types, being purchased from its woods Bel's instrument manufacturing of Haimen City has
Limit company；EPICS XL-4 type fluidic cell systems, are purchased from Beckman Coulte companies of Britain；H-7500 transmission electron microscopies
Mirror, is purchased from HIT；ULTRACUTR ultramicrotome, is purchased from Lycra company of German Austria；Constant temperature Delevoping cartridge is risen,
It is purchased from Sanming, Fujian Province city Zhi Shan photographic equipments company；DB-3 stainless steel electric hot plates, are purchased from Fuhua Instrument Ltd. of Jintan City；Electricity
Hot constant incubator, is purchased from Wuhan elite Science ＆ Teaching Instrument Co., Ltd；Water isolation type electro-heating standing-temperature cultivator, is purchased from Shanghai leap doctor
Treat Instruments, Inc.
3rd, the preparation of agents useful for same：
1) general reagent used by cell is prepared
RPMI-1640 culture mediums：The bag of 1640 powder packaging one of purchase is taken, adds white powder HEPES 2g and analysis pure
NaHCO32g, is dissolved with the tri-distilled water after 800mL autoclavings and mixed, and is subsequently adding the 100U/mL of the 5mL for having prepared
Penicillin and streptomysin, gently shake up, and after thoroughly dissolving, then constant volume is to 1000mL, using 0.22 μm of miillpore filter,
Carry out vacuum filter degerming, then packing is placed on 4 DEG C of Refrigerator stores, and 10% new fetal calf serum is added when using.
DMEM culture mediums：Take the bag of DMEM powder packagings one of purchase, add white powder HEPES 2g and analytically pure
NaHCO31.7g, then its preparing and packaging method is identical with RPMI-1640 culture medium formulation operations steps.
Phosphate buffer solution (PBS)：NaCl 4.00g, KH are accurately weighed using electronic balance2PO40.10g,
Na2HPO41.45g, KCl 0.10g, then the distilled water 450mL of stand-by storage dissolved, arrived using distilled water constant volume afterwards
500ml, is put into high-pressure sterilizing pot, and sterilize 30min at a high temperature of 121 DEG C, after cooling down, places into 4 DEG C of ice
Case is preserved and used.
3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides (MTT) solution：Electronic balance is accurately weighed
The MTT pulvis of 0.25g, is added in the PBS of 50mL, fully dissolving, until liquid is in limpid yellow, occurs without precipitation.Make
With 0.22 μm of miillpore filter, it is degerming to carry out vacuum filter, is put into 4 DEG C of refrigerators after packing is good and keeps in dark place.
Trypsin solution (0.25%)：First pancreatin 1.00g, the PBS that addition has been prepared are weighed using clean pan paper
Middle dissolving, constant volume to 400mL, using 0.22 μm of filter membrane, carries out filtration sterilization, then dispenses, and is placed on -20 DEG C of preservations.
Lead citrate solution：The accurate plumbi nitras for weighing 0.133g, the Sodium Citrate, usp, Dihydrate Powder of 0.176g is added to 3mL's
In distilled water, mixed with forced oscillation, until generating milky lead citrate suspension, add the NaOH 0.8mL of 1N,
Make solution transparent at once, if opaque, should prepare again, last constant volume to 5mL.This solution is now with the current, should try one's best reduction with
The contact of air.
AO solution：The accurate AO powder for weighing 0.5mg, adds the PBS of 500 μ L to dissolve complete under light protected environment, prepares
It is the AO storing solutions of 1mg/mL into initial concentration, masking foil is wrapped standby in 4 DEG C of Refrigerator stores.
2) Western blotting agents useful for same is prepared
30% acrylamide：The acrylamide of 29g and first-bis- fork acryloyls of 1g are weighed using clean pan paper
Amine.The distilled water mixing of 75mL is added, heating for dissolving in 37 DEG C of water-bath is placed on, then constant volume uses 0.22 μm of hole to 100mL
The membrane filtration in footpath, is placed in brown reagent bottle to refrigerate and keeps in dark place.
1.0mol/LTris-HCl(pH6.8)：The Tris for weighing 12.11g is dissolved in the distilled water of about 45mL, is used
45mL 1mol/LHCl regulation pH to 6.8, then plus distilled water dilution constant volume to arrive 100mL.4 DEG C are placed on after being filtered
Preserved in refrigerator.
1.5mol/LTris-HCl(pH8.8)：The Tris for weighing 18.15g is dissolved in the distilled water of about 45mL, is used
The 1mol/L HCl of 45mL adjust pH to 8.8, plus distilled water to dilute constant volume to 100mL.It is placed on after being filtered in 4 DEG C of refrigerators and is protected
Deposit.It is noted that both buffer solutions must all be prepared using Tris alkali, then corresponding pH value is adjusted with HCl.
10% ammonium persulfate (AP)：The ammonium persulfate that electronic balance weighs 0.25g is dissolved in the distilled water of 2.5mL, and every 500
μ L packing one is managed, and is then placed on -20 DEG C of freezen protectives.
10% dodecyl sodium sulfate (SDS)：The SDS of 10g electrophoretypes is added in the distilled water of 90mL, then at 37 DEG C
Water-bath heating for dissolving, adds distilled water constant volume to 100mL, and room temperature preservation is placed on after packing.
5 × Tris- glycine running buffers：Weigh the glycine of the Tris base and 47g of 7.55g, plus distilled water
400mL thoroughly after dissolving well, adds the 10%SDS of 25mL, finally by solution constant volume to 500mL.
5×TBS(pH8.0)：The NaCl for weighing the tris base and 21.925g of 3.025g is dissolved in the distilled water of 400mL
In, after treating fully to dissolve, then pH is adjusted to 8.0 with HCl, last constant volume to 500mL.
Transferring film buffer solution：It is accurate to weigh glycine 14.4g, tris 2.42g, add the distilled water of 700mL thoroughly to dissolve,
Absolute methanol 200mL is then slowly added into, adds distilled water constant volume to 1000mL after mixing again, sealing is preserved, and prevents absolute methanol
Volatilization.
TBST：To the Tween-20 that 0.05% is added in the TBS solution prepared.
Confining liquid：5% skimmed milk power is prepared using TBST solution, is well mixed.
Kao Mosi light blue dyeing liquors：The methyl alcohol of 45mL, the distilled water of 45mL, the glacial acetic acid of 10mL after being well mixed, is added
The coomassie gram light blue of 0.25g, fully dissolving, are placed on preservation in brown reagent bottle.
12% separation gel：Distilled water 1.9mL, the 1.5M Tris-HCl of 30% acrylamide 3.5mL, 3.42mL,
The 10%AP of the 10%SDS of 0.09mL, 0.09mL, the TEMED of 3.6 μ L, cumulative volume 9mL.
10% separation gel：Distilled water 2.33mL, the 1.5M Tris-HCl of 30% acrylamide 3.07mL, 3.42mL,
The 10%AP of the 10%SDS of 0.09mL, 0.09mL, the TEMED of 3.6 μ L, cumulative volume 9mL.
6% separation gel：Distilled water 3.5mL, the 1.5M Tris-HCl of 30% acrylamide 1.9mL, 3.42mL,
The 10%AP of the 10%SDS of 0.09mL, 0.09mL, the TEMED of 7.2 μ l, cumulative volume 9mL.
Concentration glue：The 1.0M Tris-HCl of distilled water 4.1mL, 30% acrylamide 1mL, 0.75mL, the 10% of 60 μ L
SDS, the 10%AP of 60 μ L, the TEMED of 6 μ L, cumulative volume 6mL.
0.02% Sodium azide：Preparing Sodium azide needs wearing gloves and mouth mask, weighs Sodium azide and is dissolved in PBS solution, then
It is placed on 4 DEG C of refrigerator storages standby.
Embodiment 1：
3- hydrogenates loose Siberian cocklebur acid cyanogen ethyl ester, and its systematic naming method is：(2R) -2- ((3S, 10S, 13R, 14R, 17R) -3- hydroxyls -
Tetrahydrochysene -1H- the rings penta [a] of 4,4,10,13,14- pentamethyls -2,3,4,5,6,7,10,11,12,13,14,15,16,17- ten phenanthrene -
17- yls) -6- methyl hept- 5- olefin(e) acid cyanogen methyl esters；Its structural formula is：
The synthetic method of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is：
1) the equimolar loose Siberian cocklebur acid B of 3- hydrogenations, bromoacetonitrile, potassium carbonate are added in reaction bulb, and adds appropriate acetonitrile to make
It is solvent, after the completion of 85 DEG C of stirring reaction 2h, TCL detection reactions, is spin-dried for solvent, adds water and ethyl acetate, separating and extracting
Three times, ethyl acetate is reclaimed, obtain solids；
2) by the solid method loading of solids, 200-300 mesh normal-phase silica gel column chromatography is separated, with petroleum ether：Ethyl acetate=15:
1 (volume ratio) is mobile phase, obtains white solid powder for 3- hydrogenates pine Siberian cocklebur acid cyanogen ethyl ester.
The loose Siberian cocklebur acid cyanogen ethyl ester Structural Identification hydrogen spectrum of 3- hydrogenations for obtaining is shown in Fig. 1.
1H-NMR(400MHz,CDCl3)δ：0.72(3H,s),0.81(3H,s),0.88(3H,s),0.96(3H,s),
1.02 (3H, s), 1.06-1,38 (6H, m), 1.58 (3H, s), 1.68 (3H, s), 1.53~1.88 (8H, m) 1.91-2.09
(9H, m), 2.38 (1H, m), 3.23 (1H, dd, J=4Hz, 8Hz), 4.71 (2H, m), 5.04 (1H, m).The compound is through core
Magnetic resonance device test, hydrogen spectrum (1H-NMR) high field region occur δ 0.72 (3H, s), 0.81 (3H, s), 0.88 (3H, s), 0.96
(3H, s), 1.02 (3H, s), 1.06-1,38 (6H, m), 1.58 (3H, s), 1.68 (3H, methyl proton signal peak s), respectively
For 3- hydrogenates seven methyl proton signals in loose Siberian cocklebur acid cyanogen ethyl ester.There is a series of proton letter in high field region δ 1.5~2.2
Number, thus it is speculated that the proton signal of loose Siberian cocklebur acid B is hydrogenated for parent nucleus three.Chemical shift is in δ 2.38 (1H, proton signal m), thus it is speculated that for double
The proton signal of key.Chemical shift is in 4.71 (2H, proton signal m), thus it is speculated that to connect the proton signal of the methylene of cyano group
Peak.Chemical shift is in δ 5.04 (1H, proton signal m), thus it is speculated that the diagnostic protons signal of hydroxyl in the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations
Peak.From nucleus magnetic hydrogen spectrum data analysis, the carboxyl of cyanoethyl and the loose Siberian cocklebur acid B of three hydrogenations is combined into 3- and hydrogenates with the pattern of ester bond
Loose Siberian cocklebur acid cyanogen ethyl ester.
The preparation of the loose Siberian cocklebur acid cyanogen ethyl ester storing solution of 3- hydrogenations：The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is accurately weighed using pan paper
1.5mg, accurately adds DMSO whirlpools to shake 30s with liquid-transfering gun, and the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations for being finally made 10mM is initially dense
Degree.When using, then it is diluted with the culture medium containing serum.
Embodiment 2：
First, on the basis of embodiment 1, the resulting loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is specifically tested, specifically
Method is as follows：
1st, cell culture
Human stomach cancer cell line HGC-27, HepG2 cell lines, human lung carcinoma cell line A549, Breast cancer lines
MDA-MB-231, Human carcinoma of prostate cell line PC3, human nasopharyngeal carcinoma cell line CNE-2, respectively with containing 10% hyclone
RPMI-1640 medium cultures, nerve cell blastoma SH-SY5Y, human colon carcinoma HT-29 cell lines, respectively with containing
The DMEM medium cultures of 10% hyclone, are placed on 37 DEG C and 5%CO2Saturated humidity incubator in carry out open individual layer
Cell culture.When observation of cell density is 80% or so under inverted microscope, passed on.During passage, old culture is first discarded
Liquid, is then washed twice with the PBS of 5mL, adds 0.25% trypsase, is placed on 37 DEG C, 5%CO21-5 points is incubated in incubator
Clock, when seeing cell rounding under the microscope, cell is blown and beaten with serum-containing media, terminates digestion, then moves cell suspension
To in corresponding centrifuge tube, it is centrifuged 5 minutes under 1300r/min, the supernatant discarded in aseptic operating platform, with trainings of the 2mL containing serum
Foster base uniformly blows and beats the cell in centrifuge tube, is made single cell suspension, takes 1/3 cell in new blake bottle, and supply culture medium
To 5mL, continue to cultivate.
2nd, cell transfecting culture
Take the logarithm the HT-29 cells in growth period, inoculation 1 × 105-5×105Trained in individual cell to 24 orifice plates for having culture medium
Hole is supported, cell density during transfection is reached 30-50%.Note：1) different vitro growth rates are different, the number of inoculating cell
Amount, cell density is needed according to cell type, incubation time, depending on experiment purpose；2) per hole, the cell quantity of inoculation should try one's best
It is identical, it is uniformly distributed cell.After siRNA is diluted, it is added in culture medium, gently mixes, it is ensured that transfection concentrations is
50nM.Culture plate is placed in 37 DEG C of CO224-96h is cultivated in incubator.SiRNA can be carried out within 24-72 hours after the completion of transfection
Silencing efficiency is detected.The level of target protein can be detected with Western-blot, siRNA silencing efficiencies are reacted.
Human colon carcinoma HT-29 cell line PTEN deletion forms and PTEN wild types, respectively with containing 10% hyclone
DMEM medium cultures, are placed on 37 degrees Celsius and 5%CO2Saturated humidity incubator in cultivated.Its digestion, resuspended, biography
Ride instead of walk suddenly with other human stomach cancer cell lines HGC-27, the cultural method of the cell line such as HepG2 cell lines is identical.
3rd, flow cytometer determines the change of the loose Siberian cocklebur acid cyanogen ethyl ester cell cycle of 3- hydrogenations
Because the detection of cell cycle is higher to determining the density requirements of cell, when density is too low, flow cytometer cannot do
Go out detection.Therefore, the Tissue Culture Flask culture HGC-27 cells of 50mL are needed to use in experiment, when cell attachment and growth cover
Lid degree reaches more than 80%, is initially added into the loose Siberian cocklebur acid cyanogen ethyl ester treatment stomach cancer cell of 3- hydrogenations, set concentration gradient be 10,
20th, 40 μM, and cell controls group is set, it should be noted that the density of cell controls group should be controlled 60% or so, prevent cell from existing
There is aging phenomenon during culture post processing, influence follow-up testing result.Then the cell in blake bottle is put into 5%CO2, 37
24h is cultivated in DEG C incubator.Time terminate when, cell dissociation is got off using the pancreatin without EDTA, and collect cell in from
The rotating speed centrifugation 5min of heart Guan Zhongyong 2000r/min, then gently dispels cell with PBS, and the machinery that cell is subject to is reduced as far as possible
Damage.It is centrifuged under same rotational speed, such cyclic washing 2 times, every group of cell concentration of adjustment is 1 × 106Individual/mL is advisable, and adds
Volume fraction is that 70% μ L of cold ethanol 500 are fixed, overnight.Then 2000 turns of lower centrifugations discard fixer, add reagent
The μ L of RNaseA 100 in box, liquid-transfering gun mixes cell, and the water-bath 30min in 37 DEG C of water-bath, is eventually adding 400 μ L's
PI level dyeings, are placed on 4 DEG C of refrigerators and 30min are reacted under the conditions of lucifuge, you can carry out upper machine testing using flow cytometer,
Red fluorescence of the record excitation wavelength at 488nm.
4th, flow cytometer determines apoptosis rate
Cellular processes and dosing method before Apoptosis detection are identical with cell cycle detection.Equally to note
Density of the meaning cell before dosing, prevents cellular control unit from having occurred and that aging phenomenon when processing.Different steps is pancreatin
After vitellophag, collect cell and 5min is centrifuged under 2000r, after PBS washs 2 times, it is necessary to adjust every group of cell concentration in 1-5
×105In the range of individual/mL.Then with the Binding Buffer suspension cells of 500 μ L, the μ L of Annexin V-FITC 5 are added to mix
After even, the Propidium Iodide (PI) for being subsequently added into 5 μ L are mixed, PI inherently a kind of nucleic acid dye liquors, and it is acted on
In that can make apoptosis late period and the cell of necrosis that color is caught through cell membrane, but complete cell can not be coloured.So
The sample that will be handled well afterwards is placed at room temperature, and lucifuge conditioned response 5 to 15min carries out machine on flow cytometer within an hour
Detection, its result is valid data.
5th, the autophagy detection after medicine function cells
Flow cytometer determines cell autophagy ratio
AO is the fluorescent dye in experimental applications, can be dyeed autophagosome in the cell that autophagy occurs.Cause
This, using flow cytometer can quantitative determination cell occur autophagy ratio.HGC-27 cells are cultivated in big blake bottle straight
Logarithmic phase is in cell, the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations of various concentrations is added and cell controls group is set, drug-treated is thin
After born of the same parents 24h, cell is collected, washed with PBS 2 times, the AO (1mg/mL) of initial concentration is then diluted to 0.1mg/ with PBS solution
After the concentration of L, add the AO solution of 10uL in each group cell (except control group), at normal temperatures lucifuge reaction 10-15min with
Afterwards, tested and analyzed with flow cytometer at once, experimentation is repeated 3 times.
Cellular morphology after the effect of transmission electron microscope observation medicine
HGC-27 cells are cultivated in the blake bottle of 50mL, treats that cell density reaches more than 60% and growth conditions are good
When, the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations that concentration is 20 μM is added, in 5%CO2, cultivated in 37 DEG C of saturated humidity incubator, and
Blank control group is set.After medicine effect 24h, using 0.25% trypsin digestion cell, cell is blown and beaten down with liquid-transfering gun gently
Come, be sure not to cause mechanical damage to cell, 10min is centrifuged with 800 rotating speed collects cell.Using cold PBS washed cells 2 times
Afterwards, the cell that will be centrifuged fixes cell pellet overnight with 2.5% glutaraldehyde -1% osmic acid of 500 μ L, notes to dispel cell,
It is slowly added into.Cell mass is taken out after overnight, sesame size is cut into blade carries out subsequent experimental.Washed with distilled water first
Wash the cell mass that cuts 2 times, each 10-15min；It is each de- with 50%, 70%, 80%, 90% ethanol and absolute ethyl alcohol successively again
Water 15min；Twice dehydration finally is carried out using 100% acetone, and ensures each 10min.Embedding medium is used after completion
With acetone 1:1 mixed liquor is impregnated with embedding sample, and embedding sample is placed into 37 DEG C of baking oven 24h, places into water isolation type electrothermostat
48h, then to embedded sample repair piece, and prepare it is ultra-thin cut (note, cut ultra-thin section need can paint just can),
Dye 10-15min with lead citrate dye liquor again afterwards, you can use electron microscopic observation ultra-thin section, and carry out the sequence number of egative film and record,
Etc. egative film to be washed, cell results figure is scanned.
6th, molecular docking calculates the binding ability of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations and mTOR
Molecular docking be based on geometry it is complementary, it is electrical it is complementary be principle, carry out booster action, aids drug using computer
With the interaction of the molecule or target spot of acceptor.This molecular docking is the selection mTOR crystal structure A chains from PDB databases
(4JT5_A), decrystallize water, additive polarity hydrogen, electric charge.Centered on wherein part P2X, set up It is right
Connect box.Ligand molecular sets up ionization state under pH 7.4 and does energy minimization and processes.Using Glide SP methods
Molecular docking is carried out, the binding ability of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations and mTOR is calculated.With the score value of Gscore represent medicine with
The binding ability of acceptor or target spot is strong and weak, and the value of Gscore is more negative, represents that both binding abilities are stronger, interacts stronger,
Conversely, then weaker.
7th, influence of the loose Siberian cocklebur acid cyanogen ethyl ester of Western blotting detections 3- hydrogenations to correlative protein expression
Collect protein sample
HGC-27 cells are seeded in 75cm2Maxicell blake bottle in, when cell attachment well-grown and cell is close
When spending high, the drug concentration treatment cell of the loose Siberian cocklebur acid cyanogen basic, normal, high dosage of ethyl ester of 3- hydrogenations is added, collect each group after 24h respectively
Cell, every group of cell is washed using the PBS of 5mL, totally 2 times, after discarding cleaning solution PBS, every group of cell pyrolysis liquid of the μ L of addition 100
(50mmol/L Tris-Hcl pH8.0,5mmol/LEDTA, 1%triton X-100,150mmol/LNaCl, 1mmol
PMSF) and 1 μ L inhibitors of phosphatases (preventing phosphorylated protein dephosphorylation).Freeze thawing after mixing in preparation on ice repeatedly is split
Solution 3 times, every time 15 minutes.12000 rotating speed centrifugation 10-15min, takes its supernatant, is made of BCA determination of protein concentration kits
Go out protein standard curve figure, another one calculates the total protein content of each sample.Finally each histone is calculated, it is quantitative to together
One concentration, fixed measured albumen adds 2 × albumen sample-loading buffer, and 5-7min is boiled in boiling water, is dispensed into small-sized EP pipes,
It is put into -80 DEG C of Refrigerator stores standby.
With glue
The clean gauze of glass plate used will be tested first to clean, and is rinsed 2 times with distilled water, it is standby after drying.
According to the installation instructions of instrument, glass plate is installed, hunted leak with distilled water to anhydrous and spilt.According to needed for electrophoresis
The size of molecular weight of albumen, selects suitable gel strength.According to agent prescription, prepare separation gel and be placed in clean beaker and stir
Mix uniform, then slowly poured into liquid with liquid-transfering gun between two glass plates, (attention can not pour into bubble, if there is bubble,
Bubble is driven out of rapidly with discarded sample introduction needle), separation gel stops filling sample after pouring into 4mL, and distilled water sealing is used immediately.Need to slowly add
Water, until upper horizontal with glass plate, it is too urgent to be sure not to add water, and overflows separation gel.Separation gel is placed on 37 DEG C of baking oven
In, horizontal rest 30-45min.Whether observation glue there is a refractive power line with the interface of water, has, and represents that separation gel has solidified
It is good, the water of closing can be outwelled, then unnecessary water is blotted with filter paper, and the concentration glue for having prepared and having mixed finally is poured into, comb is plugged,
Be can be used after solidification 1h at room temperature.
Loading
After concentration gelling is solid good, correctly be put into gel in electrophoresis tank by extraction comb that can be gently, pours into running buffer
Liquid, notices that electrophoresis liquid answers outside to pour into 2/3 volume, and inner side should fill.Wait loading again after 30min, the time place it is more long more
Be conducive to the formation of plastic structure, because when the gelling being observed visually is solid, its internal structure is not formed also.Will before loading
Each protein sample is placed on room-temperature dissolution, is placed on oscillator and is well mixed or boiling water water-bath 5min again, then using micro
Sample introduction needle is loaded to middle swimming lane in equal volume, and remaining swimming lane is then filled with 1 × Loading buffer of same volume.
Marker swimming lanes need to supplement 1 × Loading buffer to identical volume on the basis of Marker applied sample amounts.
Electrophoresis
After end of the sample, the both positive and negative polarity of power supply is connected according to wiring rules, concentration voltage 80v has first been set, waited band electrophoresis
During to the interface for concentrating glue and separation gel, voltage is transferred into 120v carries out band separation, then electrophoresis 1h or so, with Marker
It is reference, after electrophoresis to all of band is uniformly separated, you can stop electrophoresis.If required target protein molecular weight is small molecule
Amount, then should first use 60v electrophoresis, then shift to the voltage of 120v, if required albumen is large molecular weight protein, electrophoresis time is long
When, answer the compartment time to change ice cube, prevent electrophoresis time long, separation gel temperature is raised, and occurs melting phenomenon.
Transferring film
After electrophoresis terminates, the blob of viscose that will be unloaded is face-up, and required gel-tape is cut as mark using Marker, and
And in advance according to required gel size correspondingly-sized, an equal amount of pvdf membrane of clip one and filter paper two are standby.Gel strips
Band soaks in being put into the transferring film buffer solution of new preparation, and pvdf membrane is then distinguished in absolute methanol, distilled water, transferring film buffer solution successively
Immersion 10s, 5min, 10min, filter paper then soak 5,10min respectively in distilled water, transferring film buffer solution.Then in sequence first
Gel-tape is placed on blackboard, then puts filter paper, gel, pvdf membrane, filter paper, gel cover plate blank.It is fitted into electrophoresis tank, blackboard
Discharge source negative pole, blank discharge source positive pole, pours into transferring film buffer solution.Electrophoresis tank is put into ready ice-water bath, electrophoresis apparatus
Stabling current is adjusted to for 200mA, electrophoresis time is advisable (except high molecular weight protein) in 30-90min, enables Marker bands complete
To on cellulose membrane, then transferring film is completed for total transfer, stops electrophoresis.Gel-tape can be placed in Coomassie brilliant blue dye liquor, dyeed
After 1h, whether detergent bar band, observation transferring film is complete.
Closing
After the completion of transferring film, one end that pvdf membrane has Marker to mark gently is gripped with tweezers, first wash 3 times with TBST, often
, then be placed on film in TBST is prepared 5% skim milk by secondary 5min, and low speed shakes up incubation 2-2.5h on shaking table.
It is incubated primary antibody
After closing terminates, 3 times × 10min of cellulose membrane first is rinsed with TBST, be subsequently placed in the primary antibody that TBST diluted and incubate
Educate in bottle, slowly shaken up overnight in 4 DEG C of refrigerators, further take out within second day and be placed on shaking table jog 1h at room temperature.
It is incubated secondary antibody
After the completion of primary antibody is incubated, tweezers gently grip one end that pvdf membrane has Marker to mark, and wash 3 times with TBST, often
, then be placed on pvdf membrane in the two corresponding anti-solution that TBST has diluted by secondary 5-10min, room temperature jog 1 hour, during timer.
Washing
Secondary antibody is incubated after finishing, and cellulose membrane is then washed with TBST, is washed three times, each 10min.
Darkroom exposes
The preceding kit instrument used of darkroom exposure is got out, the pvdf membrane for completing will be first incubated with tweezers and be gently taken out,
Water unnecessary on film is blotted with paper is considered.In camera obscura, the luminescent solution that dropwise addition has been prepared (press by A liquid and B liquid for pvdf membrane placing flat
1:1 mixing), and unnecessary luminescent solution is siphoned away with filter paper.Wait pvdf membrane green fluorescence occur under dark room conditions, wait institute
Untill thering is fluorescence to occur.Pvdf membrane is covered into ready very thin overlay, sizable Kodak's glue is cut
Piece, covers on film, immediately closes off camera obscura, if fluorescence is weak to wait a few minutes more, if fluorescence is strong, at most waits 1min.Adapt to
Time moderately exposure and its it is important, after end exposure, film is placed on the 1-4min that develops in developer solution, place into clear water
Rinsing is returned, fixing 1-4min in fixing solution is finally placed on.Pvdf membrane dries in the cool after need to being cleaned with water.Film is scanned afterwards
, band can carry out quantitative analysis with gel image analysis software I mage J.Ink, and statistics compares.
8th, protective effect of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to nerve cell SH-SY5Y
Cytotoxicity of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to SH-SY5Y
Take the logarithm growth period SH-SY5Y cells with 5 × 104The concentration of individual/mL is inoculated in 96 orifice plates, 100 μ L/ holes, patch
After wall growth 12h, the various concentrations (80,40,20,10,5,2.5,1.25 μM) of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations are added, each is dense
Degree 3 multiple holes of setting, after culture 48h, mtt assay detects the inhibiting rate of cell, and calculates the corresponding IC50 values of medicine.
Protective effect of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to SH-SY5Y cells
SH-SY5Y cells are divided into 2 groups：Blank group and model group, the SH-SY5Y cells in growth period of taking the logarithm are with 5 × 104
The concentration of individual/ml is inoculated in 96 orifice plates, and 100 μ L/ holes discard old culture medium after culture 12h, model group adds various concentrations
H2O2Solution, H2O2Concentration is 1200 μM, 1100 μM, 1050 μM, 1000 μM, 950 μM, 900 μM, 800 μM, 750 μM, 700 μM.
Blank group adds DMEM culture mediums, finally ensures 200 μ L/ holes, continues to cultivate 12h, MTT colorimetric determination cell survival rates.Choosing
Taking can cause the H2O2 concentration of 40%~50%SH-SY5Y cell deaths to process cell, set up model of oxidative.
Take the logarithm growth period SH-SY5Y cells with 5 × 104The concentration of individual/mL is inoculated in 96 orifice plates, is abandoned after culture 12h
Old culture medium is removed, model group adds 1100 μM of H2O2Solution, blank group adds DMEM culture mediums, finally ensures 200 μ L/ holes, after
Continuous culture 12h, then discards old culture medium, sets blank group, model group, positive group (Verapamils of 50 μ g/mL), dosing group
(corresponding 5 μM, 2 μM, 1 μM of the concentration of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations), ensures per hole 200uL.After culture 6 hours, add per hole
The MTT of 10uL.It is measured later within 4 hours.
The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is to the study of AD rats and the influence (zoopery) of memory function
Alzheimer disease (AD) is a kind of insidious onset and carries out the nervous system degenerative disease of sexual development, clinically
With progressive memory disorders, aphasia, appraxia, agnosia, visual space infringement, perform function obstacle and personality and behavior change as special
Levy.The rat for receiving injection in A β hippocampus is relatively classical AD animal models, and the pathology mistake of AD can be preferably simulated in vivo
Journey, compared with other experimental models, the Development process of more practical reaction AD.
A β 1-40 injections induction AD experimental models in hippocampus：Each group SD rats use the intraperitoneal note of 1% yellow Jackets
Anesthesia is penetrated, AD model groups, the loose Siberian cocklebur acid cyanogen ethyl ester medicine group of 3- hydrogenations slowly at the uniform velocity inject A β 1-401 in bilateral hippocampus CAI areas
μ L (10mg/mL), let the acupuncture needle remain at a certain point 5min, control group are then injected normal saline, are sewed up a wound after the withdraw of the needle.
Choose 32 SD rats and be randomly divided into 4 groups, every group 8.Respectively control group, AD model groups, the loose Siberian cocklebur acid of 3- hydrogenations
Cyanogen ethyl ester medicine group (180mg/kg).Post operation, the daily gavage of dosing group once, 20 days altogether, gave by control group and AD model groups
Give normal saline.Since the 17th day, using Morris water mazes test assessment learning and memory in rats ability, altogether 4 days.
According to Morris water mazes operating instruction assessment test rat learning and memory ability.Institute refers to using water maze parameter
It is designated as：Round pool, diameter 180cm, 55cm high, inwall black sets depth of water 30cm, round platform is placed at pool wall 35cm (high
30cm, diameter 10cm).Experiment lasts 4 days, and experiment the previous day allows rat free swimming 2min, the daily upper and lower noon from first day
Each training 4 times.During training, place of entry is randomly selected, and rat is put into pond towards pool wall, and record each group rat finds platform simultaneously
Climb up used by platform the time (seek platform incubation period), the adjacent training time twice is at intervals of 1min.If rat is not looked in 2min
To platform, platform is artificially caused, be designated as 2min.After 4 day training time terminated, boiling water lower platform is removed, in same place by rat
It is put into pond, surveys in 2min, rat, with total time ratio, is recorded across original platform in the original platform quadrant area activity time
The number of times of position.
9th, the hypoglycemic effect of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations
Hypoglycemic effect of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to HepG2 cells
The HepG2 cells of 1640 culture mediums containing 10% hyclone are inoculated in 96 well culture plates, cell attachment 24h
Afterwards, while to the insulin that different gradient concentrations are added in hole.Experiment packet：Insulin medicament is divided into 5 groups, respectively pancreas islet
Plain 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, separately set every group of 6 multiple holes of blank control group.
Each concentration insulin action abandons old culture medium in after HepG2 cells 24h, cell is slowly cleaned with PBS three times, plus
1640 (serum-free) culture mediums stimulate 20min, abandon culture medium, cell are slowly cleaned with PBS three times, plus containing the training of 1% hyclone
After supporting base (containing each concentration insulin) culture 24h, supernatant sugared content is measured respectively by glucose kit explanation.Observation
The insulin resistant concentration of HepG2 cells.
1640 cultures containing 10% hyclone are inoculated in based in 96 well culture plates, after cell attachment 24h, old culture is abandoned
Base, cell is slowly cleaned with PBS three times, and (serum-free) culture medium stimulates 20min plus 1640, abandons culture medium, is slowly cleaned with PBS
Cell three times, plus containing 1% hyclone culture medium (medicine of insulin and each concentration containing 10-7M) the loose Siberian cocklebur acid cyanogen second of 3- hydrogenations
Ester drug concentration is respectively 80 μM, 40 μM, 20 μM, 10 μM, after 5 μM of culture 24h, is illustrated respectively to supernatant by glucose kit
Sugared content is measured.
The hypoglycemic effect (zoopery) of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations
The foundation of diabetic mouse model：Fed 1 week from male mouse of kunming adaptability, high glucose and high fat feed (feed
Composition：10% lard, 20% sucrose, 5% yolk, 65% conventional feed) to feed 8 weeks, water 12h is can't help in fasting, with 100mg/kg
The alloxan aqueous solution of intraperitoneal injection 2%, mouse continues to feed high glucose and high fat feed, after 72h (fasting 12h), after mouse orbit
Veniplex takes blood and determines blood glucose value, chooses the mouse of fasting blood-glucose (FBG) ＞ 11.1Mm as diabetic mice.
Choose normal mouse 8 and be only used as Normal group, while the successful diabetic mice 32 of modeling is chosen, it is random equal
It is divided into 4 groups, is divided into model control group, melbine positive drug control group (200mg/kg),
The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is low, high dose group (120,240mg/kg).The daily gavage of positive drug, administration group is given
Medicine 1 time, normal group and model group give isometric 0.5%CMC-Na solution, continuous gavage 4 weeks.During experiment, except normal right
Conventional feed being given according to group and feeding outer, remaining each group is with high glucose and high fat forage feed.
10th, statistical method
Statistics and analysis are carried out to data using the softwares of SPSS 13.0, data all use mean ± standard deviationShape
Formula is represented.Difference single factor test variance between group is analyzed, and P is compared with control group<0.05 has aobvious for both data differences
Work property result, P is compared with control group<0.01 has pole significant result for both data differences.
2nd, experimental result：
1st, inhibitory action of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to kinds of tumor cells
Using 5~80 μM of 3- hydrogenate loose Siberian cocklebur acid cyanogen ethyl ester process HGC-27, HepG2, A549, MDA-MB-231, PC3,
After CNE-2 cells 24h, MTT detections 3- hydrogenates inhibitory action of the loose Siberian cocklebur acid cyanogen ethyl ester to each cell, and calculates IC50 values, as a result
See Tables 1 and 2.
The loose Siberian cocklebur acid cyanogen ethyl ester of the 3- of table 1 hydrogenations to the inhibitory action of different tumour cells (n=3,)
The 3- of table 2 hydrogenates the inhibitory action of loose Siberian cocklebur acid cyanogen ethyl ester and taxol to HGC-27 cells
2nd, the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations promotes cell autophagy and produces pharmacological action
Cell cycle influences of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to HGC-27
Loose Siberian cocklebur acid cyanogen ethyl ester is hydrogenated using 10 (B), 20 (C), 40 (D) μM of 3- and process HGC-27 cell 24h, and set thin
Born of the same parents' control group (A).After PI dyeing, the loose Siberian cocklebur acid cyanogen ethyl ester of detection 3- hydrogenations is produced to the cell cycle of HGC-27 cells
Influence.Found from the result of flow cytometry analysis, the loose Siberian cocklebur acid cyanogen ethyl ester effect HGC-27 of 3- hydrogenations of basic, normal, high dosage group
After cell, the cell distribution of corresponding G0/G1 phases be respectively (47.5 ± 4.7) %, (53.1 ± 3.3) %, (58.6 ±
3.8) %, compares with cell controls group (41.5 ± 4.1) %, and the TAB of middle dose group makes the retardance of stomach cancer cell conspicuousness in G0/
G1 phases (P<0.05), high dose group can pole conspicuousness blocks cellular in G0/G1 phases (P<0.01).Its analysis result such as institute of table 3
Show, illustrate that the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations makes stomach cancer cell HGC-27 be blocked in the G0/G1 phases, so as to suppress stomach cancer cell HGC-27
Growth and propagation.
The loose Siberian cocklebur acid cyanogen ethyl ester of the 3- of table 3 hydrogenations to the HGC-27 cell cycles influence (n=3,)
Note：*The P compared with control group<0.05；**The P compared with control group<0.01.
Influence of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to HGC-27 Apoptosis
Loose Siberian cocklebur acid cyanogen ethyl ester is hydrogenated using the 3- of 10 (B), 20 (C), 40 μM (D) and process HGC-27 cell 24h, and set
Cell controls group (A).Apoptosis of the loose Siberian cocklebur acid cyanogen ethyl ester to HGC-27 cells are hydrogenated using flow cytomery 3-, specifically
See Fig. 2；Wherein A:Control group；B：Loose Siberian cocklebur acid cyanogen ethyl ester (10 μM) of 3- hydrogenations；C：Loose Siberian cocklebur acid cyanogen ethyl ester (20 μM) of 3- hydrogenations；D：3-
Loose Siberian cocklebur acid cyanogen ethyl ester (40 μM) is hydrogenated, 4 quadrants in experiment are respectively：Upper left (AV ﹣, PI ﹢)；Upper right (AV ﹢, PI ﹢)；Lower-left
(AV ﹣, PI ﹣)；Bottom right (AV ﹢, PI ﹣).AV ﹢ dyeing represents early apoptosis, and PI ﹢ dyeing represents necrosis.Experimental result discovery, 3- hydrogen
Changing loose Siberian cocklebur acid cyanogen ethyl ester does not have to make stomach cancer HGC-27 cell early apoptosis phenomenons, finds the ratio of non-viable non-apoptotic cell with medicine agent
Amount rising is incrementally increased.The loose Siberian cocklebur acid cyanogen ethyl ester of this result explanation 3- hydrogenations makes the dead modes of stomach cancer HGC-27 may not be logical
Cross apoptotic pathways.
Influence of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to HGC-27 cell autophagies
Cell subclones after the loose Siberian cocklebur acid cyanogen ethyl ester effect of transmission electron microscope detection 3- hydrogenations, are specifically shown in Fig. 3, wherein a：
Control group (× 6000) b/c：Autophagic vacuole (× 6000) d:Autophagosome submicroscopic structure (× 15000)：
In the HGC-27 cells of exponential phase, cell controls group and loose Siberian cocklebur acid cyanogen ethyl ester dosing group (20 μ of 3- hydrogenations are set
M), after treatment cell 24h, cell is collected, fixes, is dehydrated, is impregnated with, embeds, prepares ultra-thin section, finally complete dyeing, transmitted
Electron microscopic observation ultra-thin section, Taking Pictures recording result.Electronic Speculum result shows the cell of control group, and nucleus is big and complete, organelle
Marshalling, without missing, the edge of cell membrane is also high-visible.Conversely, the stomach cancer after the loose Siberian cocklebur acid cyanogen ethyl ester effect of 3- hydrogenations
There is defect or missing in cell HGC-27, nucleus, most organelle is all damaged, the autophagy of substantial amounts of bilateral membrane stage
Bubble, autophagosome occur, and more have substantial amounts of autophagy vacuole also containing the digestion material of residual in autophagosome.Experimental result table
There is autophagy in the bright loose Siberian cocklebur acid cyanogen ethyl ester induction stomach cancer cell HGC-27 of 3- hydrogenations.
AO decoration methods detect cell autophagy ratio, are specifically shown in Fig. 4, and the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is to HGC-27 cell autophagies
The influence of ratio, wherein A:Control group；B：Loose Siberian cocklebur acid cyanogen ethyl ester (10 μM) of 3- hydrogenations；C：Loose Siberian cocklebur acid cyanogen ethyl ester (20 μ of 3- hydrogenations
M)；D：Loose Siberian cocklebur acid cyanogen ethyl ester (40 μM) of 3- hydrogenations.
The corpusculum that autophagy occurs can be dyeed by AO, then can detect its cell for dyeing using flow cytometer.Therefore, profit
The ratio that can be quantified and detect cell autophagy is dyeed with AO.Loose Siberian cocklebur acid cyanogen ethyl ester is hydrogenated using the 3- of basic, normal, high dosage respectively to process
Cell is collected after HGC-27 cells 24h, and sets blank control group.Through flow cytomery, as a result find, 10,20,40 μM
After the loose Siberian cocklebur acid cyanogen ethyl ester treatment cell of 3- hydrogenations, 10 μM of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations does not have to make autophagy ratio substantially change
Become, and the loose Siberian cocklebur acid cyanogen ethyl ester of 20,40 μM of 3- hydrogenations makes the autophagy ratio of cell increase to 3.49 ± 2.37%, 43.85 respectively
± 7.09%, compare with cell controls group (0.58 ± 0.56%), cell autophagy ratio gradually rises, and high dose group 3- hydrogen
The autophagy ratio that loose Siberian cocklebur acid cyanogen ethyl ester (40 μM) of change causes compares the difference (P with pole conspicuousness with blank group<0.01).
The detection of autophagy GAP-associated protein GAP after the loose Siberian cocklebur acid cyanogen ethyl ester effect HGC-27 cells of 3- hydrogenations：
Concentration is 10,20,40 μM of the loose Siberian cocklebur acid cyanogen ethyl ester treatment HGC-27 cells of 3- hydrogenations, and sets cell
Control group, cell extraction total protein is collected after effect 24h.The testing result of Western blotting is shown in Table 4, the pass of autophagy
There is obvious increase, 20,40 μM of the loose Siberian cocklebur acid of 3- hydrogenations with the rising of drug concentration in the expression of key modulin Beclin-1
Cyanogen ethyl ester makes the expression of Beclin-1 extremely significant rising occur；LC3 is also to participate in the important gene that autophagy occurs.Testing result
There is the reduction of conspicuousness in display, the ratio of I/LC3 of LC3 II, illustrate LC3 I with 3- hydrogenate the rising of loose Siberian cocklebur acid cyanogen ethyl ester concentration by
Gradually converted to LC3 II, and basic, normal, high dosage all has extremely significant difference.Western blotting results show that 3- is hydrogenated
Loose Siberian cocklebur acid cyanogen ethyl ester causes autophagy key protein to there occurs change.
Influence that the loose Siberian cocklebur acid cyanogen ethyl ester of the 3- of table 4 hydrogenations is expressed autophagy proteins (n=3,)
Note：Compared with blank group*P<0.05；**P<0.01
3rd, the mechanism that the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations promotes cell autophagy is：The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is PI3K/
MTOR double inhibitors.
Influence of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to PI3K/AKT/mTOR signal paths
Molecular docking experimental result：
MTOR crystal structure A chains (4JT5_A) is chosen from PDB databases, decrystallize water, additive polarity hydrogen, electric charge.With it
Centered on middle part P2X, set upDocking box.Ligand molecular sets up the ionization under pH 7.4
State simultaneously does energy minimization treatment.Molecular docking is carried out using Glide SP methods, molecular docking binding mode is as follows：
Part is located at Gln2161, Ile2163, Thr2164, Ser2165, Pro2169, Leu2185, Lys2187,
Leu2192, Asp2195, Met2199, Ile2237, Trp2239, Cys2243, Asp2244, Thr2245, Hid2247,
In the active pocket of the amino acid residues such as Arg2251, Ser2342, Met2345, Ile2356, Asp2357, Phe2358 composition.
There is hydrogen bond action between ketonic oxygen and Lys2187 in the carboxyl of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations.Loose Siberian cocklebur acid cyanogen ethyl ester is hydrogenated with 3-
The Gscore of docking is -5.36.From the point of view of Computer simulation results more than, the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is illustrated in theory
There are good affinity and interaction with mTOR.The molecular docking result figure 5 of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations and mTOR.
Influence of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to key protein expression in PI3K/AKT/mTOR signal paths：
The loose Siberian cocklebur acid cyanogen ethyl ester concentration of 3- hydrogenations is 20 μM for the treatment of HGC-27 cells, and sets cell controls group, after effect 24h
Collect cell and extract total protein, carry out protein quantification.Western blotting have detected the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations and make
After with cell 24h, the expression of P-PI3K, P-mTOR and signal path downstream P-p70S6K albumen in signal path.It is real
Result is tested to show, after the loose Siberian cocklebur acid cyanogen ethyl ester effect HGC-27 cells 24h of 3- hydrogenations, P-PI3K, P-mTOR, P-p70S6K albumen table
Up to the downward for all occurring in that conspicuousness, with blank group contrast the difference for having pole conspicuousness.Specifically it is shown in Table 5.
The loose Siberian cocklebur acid cyanogen ethyl ester of the 3- of table 5 hydrogenations to different protein expressions influence (n=3,)
Note：Compared with blank group*P<0.05；**P<0.01
4th, the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations suppresses mTOR has antitumaous effect, and good to PTEN deletion forms tumor effect.
The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is to PTEN deletion forms and the inhibitory action of the HT-29 cells of wild type
PTEN plays the effect of key as a kind of tumor suppressor in PI3K/AKT/mTOR signal paths.PTEN's lacks
Lose or lose, PI3K/AKT/mTOR signal pathways can be activated.In clinical trial, research finds, patients with gastric cancer group
The middle phenomenon for a large amount of PTEN missings occur is knitted, and in healthy population, PTEN is positive expression, therefore, PTEN and PI3K/
There may be close association between AKT/mTOR signal paths.
The PTEN wild types and PTEN deletion form HT-29 cells in growth period of taking the logarithm is inoculated in 96 orifice plates, culture 12h with
Afterwards, the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is added, drug concentration is (80,40,20,10,5,2.5,1.25 μM), after culture 48h, MTT
Method detects the inhibiting rate of cell, calculates the corresponding IC50 values of medicine.The loose Siberian cocklebur acid cyanogen ethyl ester of result display 3- hydrogenations is significantly inhibited
PTEN deletion form HT-29 cells.Specifically it is shown in Table 6.
The loose Siberian cocklebur acid cyanogen ethyl ester of the 3- of table 6 hydrogenations is to PTEN wild types and the half-inhibition concentration of deletion form HT-29 cells
(IC50) (n=3,)
5th, the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations suppresses mTOR has nerve the moving back property venereal diseases such as treatment Alzheimer's (AD)
Change is acted on.
Protective effect of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to nerve cell SH-SY5Y
Cytotoxicity of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to SH-SY5Y：
It is thin that loose Siberian cocklebur acid cyanogen ethyl ester various concentrations (80,40,20,10,5,2.5,1.25 μM) of 3- hydrogenations acts on SH-SY5Y
Born of the same parents, after culture 48h, mtt assay detects the inhibiting rate of cell, calculates the corresponding IC50 values of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations.Result shows
Show that medicine is smaller to SH-SY5Y cytotoxicities.Specifically it is shown in Table 7.
The loose Siberian cocklebur acid cyanogen ethyl ester of the 3- of table 7 hydrogenations to the cytotoxicity of SH-SY5Y (n=3,)
Protective effect of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations to SH-SY5Y cells：
Use H2O2Various concentrations process cell, set up model of oxidative.Experimental result is drawn in H2O21100 μM
Under concentration, the inhibiting rate of nerve cell reaches 42.71%.SH-SY5Y cells are grouped into blank group, model group, positive group (50
The Verapamil of μ g/mL), dosing group (5 μM, 2 μM, 1 μM of the corresponding concentration of medicine), it is ensured that per hole 200uL.Culture 6 hours with
Afterwards, the MTT of 10uL is added per hole.It is measured later within 4 hours.The result display μ g/mL Verapamil energy conspicuousnesses of positive drug 50
SH-SY5Y cells after hydrogen peroxide is damaged are improved, has protective effect to nerve cell.The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is to nerve cell
Also there is protective effect, when the loose Siberian cocklebur acid cyanogen ethyl ester concentration of 3- hydrogenations is 5 μM, show extremely significant protective effect, have at 2 μM significantly
The protective effect of property.Specifically it is shown in Table 8.
The loose Siberian cocklebur acid cyanogen ethyl ester of the 3- of table 8 hydrogenations to the protective effect of SH-SY5Y cells (n=3,)
Note：*The P ＜ 0.05 compared with model group；**The P ＜ 0.01 compared with model group
The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is to the study of AD rats and the influence (zoopery) of memory function：
Determine each group rat and seek platform incubation period in 4 days in Morris water maze laboratories, be specifically shown in Table 9, table 10, table
11.Result show all rats seek platform incubation period all show shorten trend, but AD model group rats seek platform hide
Phase is obviously prolonged compared to control group, and the platform incubation period of seeking of loose Siberian cocklebur acid cyanogen ethyl ester (180mg/kg) medicine group of 3- hydrogenations will be significantly
Less than AD model groups.Each group rat is recorded in original platform quadrant activity time and total time ratio after removing platform, and across flat
The number of times of platform.Result finds that compared with control group, AD model group search time ratios are significantly reduced, and searching times also significantly subtract
It is few；Loose Siberian cocklebur acid cyanogen ethyl ester (180mg/kg) medicine group of 3- hydrogenations has model group to compare, and search time ratio and searching times all go out
Now significantly improve, but without recovery normal value.It is numerous test result indicate that, SD rats can be caused to learn after injection in A β hippocampus
And impaired memory function, the learning and memory function that the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations can preferably improve in hippocampus caused by injection A β receives
Damage.
In table 9Morris water maze laboratories each group rat seek the preclinical change of platform (n=4,)
Note：Compared with AD model groups*P<0.05；**P<0.01；Compared with control group#P<0.05；##P<0.01
Ratio shared by each group SD rat search times of table 10 change (n=4,)
Note：Compared with AD model groups*P<0.05；**P<0.01；Compared with control group#P<0.05；##P<0.01
The each group SD rat searching times of table 11 change (n=4,)
Note：Compared with AD model groups*P<0.05；**P<0.01；Compared with control group#P<0.05；##P<0.01
6th, the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations suppresses mTOR has treatment diabetes effect.
The hypoglycemic effect of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations
The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is specifically shown in Table 12 to the hypoglycemic effect of HepG2 cells.
Choose insulin 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, separately sets blank control group, acts on HepG2 cells
After 24h, old culture medium is abandoned, cell is slowly cleaned with PBS three times, (serum-free) culture medium stimulates 20min plus 1640, abandons culture
After base, cell is slowly cleaned with PBS three times, plus (containing each concentration insulin) containing 1% hyclone culture medium cultivates 24h, by Portugal
Grape sugar kit explanation is measured to supernatant sugared content respectively.Modeling result shows the insulin resistant concentration of HepG2 cells
10-7M。
The insulin of table 12 to HepG2 grape cell sugar consumption amounts influence (n=5,)
Using same method culture HepG2 cells, plus (contain 10 containing 1% hyclone culture medium-7The insulin of M and each
The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations of concentration), the loose Siberian cocklebur acid cyanogen ethyl ester concentration of 3- hydrogenations is respectively 80 μM, 40 μM, 20 μM, 10 μM, 5 μM
After culture 24h, supernatant sugared content is measured respectively by glucose kit explanation.The loose Siberian cocklebur acid of testing result display 3- hydrogenations
Cyanogen ethyl ester pole under 40 μM of concentration significantly reduces glucose content, is specifically shown in Table 13.
The loose Siberian cocklebur acid cyanogen ethyl ester of the 3- of table 13 hydrogenations to HepG2 grape cell sugar consumption amounts influence (n=5,)
Note：*With insulin 10-7Group compares P<0.05；**With insulin 10-7Group compares P<0.01
The hypoglycemic effect (zoopery) of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations, is specifically shown in Table 14.
Centrifuging and taking serum, fasting blood sugar is determined using glucose kit.Compared with normal group, model group mouse blood sugar
Significantly raise；Compare with model group mouse, low, the equal conspicuousness of high dose group the reduction mouse blood of the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations
Sugar value.
The loose Siberian cocklebur acid cyanogen ethyl ester of the 3- of table 14 hydrogenations to blood glucose in diabetic mice influence (n=8,)
Note：Compared with model groupaP<0.05；bP<0.01；Compared with normal groupcP<0.05；dP<0.01。
Claims (9)
1. a kind of 3- hydrogenates loose Siberian cocklebur acid cyanogen second esters medicine, it is characterised in that the chemical combination containing following structural formula in such medicine
Thing：
2. 3- according to claim 1 hydrogenates loose Siberian cocklebur acid cyanogen second esters medicine, it is characterised in that：Such class medicine also includes
Pharmaceutically acceptable salt, the loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations and its derivative pharmaceutically acceptable salt, and can pharmaceutically connect
The auxiliary material or carrier received.
3. 3- according to claim 1 hydrogenates loose Siberian cocklebur acid cyanogen second esters medicine, it is characterised in that：The loose Siberian cocklebur acid cyanogen second of 3- hydrogenations
The synthetic method of ester is：
1) the equimolar loose Siberian cocklebur acid B of 3- hydrogenations, bromoacetonitrile, potassium carbonate are added in reaction bulb, and adds appropriate acetonitrile as molten
Agent, after the completion of 85 DEG C of stirring reaction 2h, TCL detection reactions, is spin-dried for solvent, adds water and ethyl acetate, separating and extracting three times,
Ethyl acetate is reclaimed, solids is obtained；
2) by the solid method loading of solids, 200-300 mesh normal-phase silica gel column chromatography is separated, with petroleum ether：Ethyl acetate volume ratio=
15:1 is mobile phase, obtains white solid powder for 3- hydrogenates pine Siberian cocklebur acid cyanogen ethyl ester.
4. the loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations according to claim 1-3 any one is in cell autophagy is promoted
Purposes.
5. the loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations according to claim 1-3 any one is in cell autophagy is promoted
Purposes, it is characterised in that：The loose Siberian cocklebur acid cyanogen ethyl ester of 3- hydrogenations is PI3K/mTOR double inhibitors.
6. the loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations according to claim 1-3 any one is preparing treatment stomach cancer, liver
Application in terms of cancer, lung cancer, prostate cancer or medicine for nasopharyngeal.
7. the loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations according to claim 1-3 any one is preparing treatment neurological
Venereal disease becomes the application in terms of medicine.
8. the loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations according to claim 1-3 any one is preparing treatment alzheimer '
Application in terms of Mo's disease medicine.
9. the loose Siberian cocklebur acid cyanogen second esters medicine of 3- hydrogenations according to claim 1-3 any one is preparing treatment Rezulin
The application in object space face.
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