CN102319240A - Application of deacetylmycoepoxydiene serving as Hsp90 inhibitor - Google Patents
Application of deacetylmycoepoxydiene serving as Hsp90 inhibitor Download PDFInfo
- Publication number
- CN102319240A CN102319240A CN 201110201969 CN201110201969A CN102319240A CN 102319240 A CN102319240 A CN 102319240A CN 201110201969 CN201110201969 CN 201110201969 CN 201110201969 A CN201110201969 A CN 201110201969A CN 102319240 A CN102319240 A CN 102319240A
- Authority
- CN
- China
- Prior art keywords
- dam
- inflammatory
- cell
- ethyl ester
- epoxy ethyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- RAIIGSANSXCLSF-QXJJFIIZSA-N C=C([C@@H]1[C@H]([C@@H](C=C2)O)OC2=O)[C@H]2O[C@@H]1C=CC=C2 Chemical compound C=C([C@@H]1[C@H]([C@@H](C=C2)O)OC2=O)[C@H]2O[C@@H]1C=CC=C2 RAIIGSANSXCLSF-QXJJFIIZSA-N 0.000 description 1
- NABSIRLJJNMREF-KILGZEEOSA-N N=C(C=C1)O[C@H]([C@H]([C@@H]2O[C@H]3C=CC=C2)C32[IH]C2)[C@@H]1O Chemical compound N=C(C=C1)O[C@H]([C@H]([C@@H]2O[C@H]3C=CC=C2)C32[IH]C2)[C@@H]1O NABSIRLJJNMREF-KILGZEEOSA-N 0.000 description 1
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides an application of deacetylmycoepoxydiene serving as an Hsp90 inhibitor, relates to a compound deacetylmycoepoxydiene, provides an application of deacetylmycoepoxydiene in preparing an Hsp90 inhibitor and an inflammation inhibitor, and particularly provides an application of deacetylmycoepoxydiene in preparing a medicament for treating inflammatory diseases mediated by inflammatory factor TNF(tumor necrosis factor)-alpha and the like. Experiments prove that the deacetylmycoepoxydiene can effectively inhibit expression of Hsp90 and inflammatory factors, such as a tumor necrosis factor (TNF-alpha), interleukin-6 (IL-6) and nitric oxide, and can inhibit the activation of a signal pathway NF-kappa B generated by a TNF-alpha induced inflammatory factor. The deacetylmycoepoxydiene can be used as an inflammatory inhibitor in preparing medicaments for treating rheumatoid arthritis, inflammatory bowel disease, neurodegenerative diseases and other inflammatory factor mediated inflammatory diseases.
Description
Technical field
The present invention relates to a kind of compound deacetylase fungal epoxy ethyl ester (Deacetylmycoepoxydiene is abbreviated as DAM), especially relate to a kind of deacetylase fungal epoxy ethyl ester as of the application of Hsp90 inhibitor in preparation inflammation inhibitor and each field of medicine.Deacetylase fungal epoxy ethyl ester has another name called the strong rhzomorph (Nanqiangmycin) in south.
Background technology
(Heat shock proteins Hsps) is molecule companion in the eukaryotic cell to heat shock protein, and its major function is the vital movements such as renaturation and the proteic migration of cell interior of participating in new protedogenous synthetic, Denatured protein.Hsp90 plays the part of important role as a member important in the Hsps family in the vital movement of cell, its content in cell accounts for 1%～2% of cell protein total amount.
Hsp90 participates in the adjusting of nearly all physiological process such as cellular metabolism, growth, growth, differentiation, death and immunity, so its active numerous disease (like neurodegenerative diseases, cardiovascular disease, viral infection and cancer etc.) unusual and human body is closely related.Inflammatory reaction is one of important component part of body immune system, and body carries out accurate regulation and control through number of ways to it.But when inflammatory reaction is uncontrollable, can cause the multiple disease of body, like rheumatoid arthritis (rtheumatoid arthriti), inflammatory bowel (Inflammatory bowel disease; IBD), neurodegenerative disease (neurodegenerative disorder)) or the like; Secular inflammatory reaction stimulates also can bring out the body canceration, these all serious threat people physical and mental health (1, Monaco, et al.; Curr Drug Targets Inflamm Allergy; 2004,3,35-42; 2, Schwartsburd et al., Cancer Metastasis Rev, 2003,22,95-102), therefore to body too drastic inflammatory reaction to control effectively be the main direction of these disease medicaments of exploitation treatment at present.Research shows; Though the symptom that inflammatory reaction showed in the above-mentioned disease is different; But the factor or the medium that mediate these inflammatory reactions are identical; Like tumor necrosis factor-alpha (Tumor necrosis factor alpha, TNF-α), interleukin-1 ' beta ' (Interleukin-1beta, IL-1 β), interleukin-6 (IL-6) etc.Nitric oxide (NO) etc. can be by abduction delivering in inflammatory reaction, thus the inflammation degree of aggravation body (3, Heller et al., Proc Natl Acad Sci USA, 1997,94,2150-2155; 4, Feldmann et al., Nat Med, 2003,9,1245-1250), therefore these inflammatory factors are suppressed will help to improve the inflammatory reaction of body.
At present, indomethacin (indometacin), aspirin nonsteroidal and-inflammatory drugs such as (aspirin) (Non-steroidal anti-inflammatory drugs, NSAIDs) be widely used for treating the relevant disease of inflammation such as rheumatoid arthritis (5, Day et al.; Med J Aust, 1988,148; 195-199), but used NSAIDs generally has side effect, can cause symptom (Lichtenberger et al. such as gastrointestinal damage and renal function be undesired after taking for a long time; Nat Med; 1995,1,154-158; Fernandez et al., Nat Med, 1995,1,602-603).Therefore, need the further chemical compound of seeking effective inflammation-inhibiting reaction, the anti-inflammatory drug that hangs down side effect for exploitation later on provides new thinking.
The applicant discloses a kind of antineoplastic compound deacetylase fungal epoxy ethyl ester and preparation method thereof in Chinese patent 200610036892.6; A kind of fermentation substrate of antineoplastic compound deacetylase fungal epoxy ethyl ester is disclosed in Chinese patent 200810071738.1 again; Also in Chinese patent 200910112353.X3, disclose a kind of method for preparing of deacetylase fungal epoxy ethyl ester, Compound D eacetylmycoepoxydiene obtains by separating in the strain Phomopsis A-1-2-3 fermented product extract.Discover that deacetylase fungal epoxy ethyl ester has better antitumor activity premenstruum.Mtt assay vitro detection anti-tumor activity obtains its IC for the Raji cell strain
50Be 12.1 μ M, but document does not report that it has physiologically actives such as anti-inflammatory.
Deacetylase fungal epoxy ethyl ester production strains A-1-2-3 is a strain Phomopsis (Phomopsis sp.); This strains separation is careless Pu Tou village, Jiulong River Estuary Fu Gong town mangrove forest ecological district Kandelia candel mangrove leaf (Yang Lishan in the Zhangzhou City, Fujian Province; The preliminary study [D] of mangrove endophyte strain biological activity and strains A-1-2-3 secondary metabolite; Xiamen: Xiamen University, 2006).Strains A-1-2-3 on 20% sea water prescription potato agar culture medium (PDA), well-grown, mycelia is pure white, does not have obvious pigment and produces, and does not produce spore, fermentation time is 14 days.
Said deacetylase fungal epoxy ethyl ester is the chemical compound that strains A-1-2-3 metabolism produces, and said strains A-1-2-3 is a strain Phomopsis (Phomopsis sp.).
Summary of the invention
The object of the present invention is to provide deacetylase fungal epoxy ethyl ester to treat by the application in the diseases associated with inflammation medicine of mediations such as inflammatory factor TNF-α in preparation in the application, particularly deacetylase fungal epoxy ethyl ester of preparation Hsp90 inhibitor and inflammation inhibitor thereof.
The chemical formula of said deacetylase fungal epoxy ethyl ester is C
14H
16O
4, molecular weight is 246, its chemistry 2-(8-methyl-9-oxa--dicyclo [4.2.1] nine-2,4-diene-7-yl) by name-6-oxygen-3, and 6-dihydro-2H-pyrone is white pinniform or bar-shaped transparent crystallization, chemical structural formula is:
Said deacetylase fungal epoxy ethyl ester can derive from fungus fermentation products or chemosynthesis.
Said deacetylase fungal epoxy ethyl ester is treated by the application in the diseases associated with inflammation medicine of mediations such as inflammatory factor TNF-α in preparation in the application, particularly deacetylase fungal epoxy ethyl ester of preparation Hsp90 inhibitor and inflammation inhibitor thereof.
Said diseases associated with inflammation comprises rheumatic arthritis, inflammatory bowel, neurodegenerative disease etc.
Through experiment confirm; Deacetylase fungal epoxy ethyl ester (is designated as the strong rhzomorph in south; DAM) expression of Hsp90 and inflammatory factor such as tumor necrosis factor (TNF-α), interleukin-6 (IL-6) and nitric oxide (NO) can be suppressed effectively, and activation can be suppressed by the signal path NF-kappaB of the inductive inflammatory factor generation of TNF-α.Can be used as inflammation inhibitor and be used to prepare the medicine by the diseases associated with inflammation of inflammatory factor mediation such as treatment rheumatic arthritis, inflammatory bowel, neurodegenerative disease.
Description of drawings
Fig. 1 is the MTT experimental result picture after DAM (the strong rhzomorph in south) acts on the RAW264.7 cell.In Fig. 1, abscissa is DAM concentration (μ mol/L), and vertical coordinate is absorbance (595nm).
Fig. 2 is the MTT experimental result picture after DAM acts on the RAW264.7 cell under the bacteria lipopolysaccharide stimulation.In Fig. 2, abscissa is DAM concentration (μ mol/L), and vertical coordinate is the absorbance of 595nm; LPS concentration is 100ng/mL.
Fig. 3 is the influence figure of DAM to proinflammatory vitamin T NF-alpha expression in the RAW264.7 cell that stimulates through bacteria lipopolysaccharide.In Fig. 3, abscissa is DAM (μ mol/L), and vertical coordinate is proinflammatory vitamin T NF-α (pg/ml); DAM concentration is that 0 group is a DMSO blank group, and other concentration group are carried out the t check with the DMSO group respectively; * represent p＜0.05, * * representes p＜0.01
Fig. 4 is the influence figure of DAM to proinflammatory factor IL-6 expression in the RAW264.7 cell that stimulates through bacteria lipopolysaccharide.In Fig. 4, abscissa is DAM concentration (μ mol/L), and vertical coordinate is proinflammatory factor IL-6 (pg/ml); DAM concentration is that 0 group is a DMSO blank group, and other concentration group are carried out the t check with the DMSO group respectively; * representes p＜0.01.
Fig. 5 is the influence figure of DAM to proinflammatory factor IL-1 β expression in the RAW264.7 cell that stimulates through bacteria lipopolysaccharide.In Fig. 5, abscissa is DAM concentration (nmol/L), and vertical coordinate is proinflammatory factor IL-1 β (pg/ml); DAM has the obvious suppression effect in the expression to proinflammatory factor IL-1 β in the RAW264.7 cell of bacteria lipopolysaccharide stimulation under 9, the 12 μ mol/L concentration; DAM concentration is that 0 group is a DMSO blank group, and other concentration group are carried out the t check with the DMSO group respectively.
Fig. 6 is the influence of DAM to short inflammatory mediator TNF-alpha expression in the RAW264.7 cell of variable concentrations bacteria lipopolysaccharide stimulation.In Fig. 6; Abscissa is the Concentraton gradient (ng/ml) of bacteria lipopolysaccharide, and vertical coordinate is that the concentration (pg/ml) of TNF-α: DAM short inflammatory mediator TNF-alpha expression in the RAW264.7 cell that under the 10 μ mol/L concentration bacteria lipopolysaccharide through variable concentrations is stimulated has the obvious suppression effect.The LPS group is carried out the t check with the LPS+DAM group respectively among the figure, and DAM concentration is 10 μ mol/L; * represent p＜0.05;
is LPS, and
is LPS+DAM.
Fig. 7 is the influence that DAM expresses short inflammatory mediator IL-6 in the RAW264.7 cell of variable concentrations bacteria lipopolysaccharide stimulation.In Fig. 7; Abscissa is the Concentraton gradient (ng/ml) of bacteria lipopolysaccharide, and vertical coordinate is that the concentration (pg/ml) of IL-6: DAM short inflammatory mediator IL-6 expression in the RAW264.7 cell that under the 10 μ mol/L concentration bacteria lipopolysaccharide through variable concentrations is stimulated has the obvious suppression effect; The LPS group is carried out the t check with the LPS+DAM group respectively among the figure, and DAM concentration is 10 μ mol/L; * representes p＜0.01;
is LPS, and
is LPS+DAM.
Fig. 8 is the influence that DAM expresses short inflammatory mediator IL-1 β in the RAW264.7 cell of variable concentrations bacteria lipopolysaccharide stimulation.In Fig. 8; Abscissa is the Concentraton gradient (ng/ml) of bacteria lipopolysaccharide, and vertical coordinate is that the concentration (pg/ml) of IL-1 β: DAM short inflammatory mediator IL-1 β expression in the RAW264.7 cell that under the 10 μ mol/L concentration bacteria lipopolysaccharide through variable concentrations is stimulated has the obvious suppression effect.The LPS group is carried out the t check with the LPS+DAM group respectively among the figure, and DAM concentration is 10 μ mol/L;
is LPS, and
is LPS+DAM.
Fig. 9 is the influence that DAM expresses NO in the RAW264.7 cell that stimulates through bacteria lipopolysaccharide.In Fig. 9, abscissa is the Concentraton gradient (μ mol/L) of DAM, and vertical coordinate is the concentration (μ mol/L) of NO, and DAM has the obvious suppression effect in the expression to NO in the RAW264.7 cell of bacteria lipopolysaccharide stimulation under 9, the 12 μ mol/L concentration; DAM concentration is that 0 group is a DMSO blank group among the figure, and other concentration group are carried out the t check with the DMSO group respectively; * represent p＜0.05, * * representes p＜0.01.
Figure 10 is the inhibitory action of deacetylase fungal epoxy ethyl ester (DAM) to nuclear factor (NF-kappaB) signal transduction pathway of inflammatory factor generation in the RAW264.7 cell.In Figure 10, show with the deacetylase fungal epoxy ethyl ester (DAM) of 1,2.5 and 5 μ g/mL and handle HeLa cell 12h, the expression of nuclear factor in cytoplasm and the nucleus (RelA) and CKIs matter I κ B thereof.β-actin is the protein contrast in the cytoplasm; PARP is the protein contrast (Figure 10) in the nucleus.
Figure 11 activates 0.5h with LPS (100ng/mL), handles RAW264.7 cell 5.5h, the expression of nuclear factor in cytoplasm and the nucleus (RelA) and CKIs matter I κ B thereof with the deacetylase fungal epoxy ethyl ester (DAM) of 1,2,4 and 6 μ g/mL.β-actin is the protein contrast in the cytoplasm; PARP is the protein contrast (Figure 10) in the nucleus.
Figure 12 activates 0.5h with inflammatory factor TNF-α (20ng/mL); Handle RAW264.7 cell 5.5h, the expression of nuclear factor in cytoplasm and the nucleus (RelA) and CKIs matter I κ B thereof with the deacetylase fungal epoxy ethyl ester (DAM) of 1,2,4 and 6 μ g/mL; β-actin is the protein contrast in the cytoplasm; PARP is the protein contrast (Figure 12) in the nucleus.
Figure 13 is the influence figure of DAM to Hsp90 in the HeLa cell and related protein expression thereof.In Figure 13, the Western blot analysis result that related protein is expressed behind the demonstration DAM effect HeLa cell 24h.Figure 13 shows the increase along with DAM concentration (1,2.5,5 μ g/mL); Client's albumen of Hsp90 (is abbreviated as Akt, IKK α, IKK β; The Akt of phosphorylation) expression downward modulation, and the expression of Hsp70 significantly raises, promptly DAM has Degradation to the Hsp90 client protein; The positive contrast of GA among Figure 13 (geldanamycin).
Figure 14, Figure 15 are the inhibitory action of DAM to the activated nuclear factor of inflammatory factor TNF-α (NF-kappaB) signal transduction pathway.In Figure 14, show with the deacetylase fungal epoxy ethyl ester (DAM) of 1,2.5 and 5 μ g/mL and handle HeLa cell 23.5h, and activate 0.5h with inflammatory factor TNF-α (20ng/mL).The expression of nuclear factor in cytoplasm and the nucleus (RelA) and CKIs matter I κ B thereof.β-actin is the protein contrast in the cytoplasm; PARP is the protein contrast (Figure 14) in the nucleus; In Fig. 8, show with the deacetylase fungal epoxy ethyl ester (DAM) of 2.5 μ g/mL and handle HeLa cell 23.5h, activate 0.5h with inflammatory factor TNF-α (20ng/mL) again.The coloration result of cell and nuclear factor RelA.Nucleus dyeing back shows blue, and RelA shows orange or red, goes into the nuclear back and presents pink (Figure 15) with nucleus colour developing stack.
Figure 16 is the influence figure of DAM to Hsp90 in the human breast carcinoma MCF-7 cell and related protein expression thereof.In Figure 16, the Western blot analysis result that related protein is expressed behind the demonstration DAM effect MCF-7 cell 6h.Figure 16 shows the increase along with DAM concentration (10,2.0,30 and 50 μ mol/L); Client's albumen (being abbreviated as Akt) of Hsp90 is under the action time of 6h; Present degraded trend, but it is not obvious to descend, the amount of the Akt of phosphorylation is obviously reduced; And the expression of Hsp70 raises, and promptly DAM has Degradation to the Hsp90 client protein.The positive contrast of GA among Figure 16 (geldanamycin).
Figure 17 is the influence figure of DAM to Hsp90 in people's pulmonary carcinoma H1299 cell and related protein expression thereof.In Figure 17, the Western blot analysis result that related protein is expressed behind the demonstration DAM effect H1299 cell 24h.Figure 17 shows along with DAM concentration (10; 2.0,30 and 50 μ mol/L) increase, client's albumen of Hsp90 (is abbreviated as Akt; IKK etc.) under the action time of 24h; Present degraded trend, and the expression of Hsp70 raises, promptly DAM also has Degradation to Hsp90 client protein in the H1299 cell.The positive contrast of GA among Figure 17 (geldanamycin).
Figure 18 influence that to be DAM express the Hsp90 among the gastric carcinoma cells BGC-823 and related protein thereof is along with time variation diagram.In Figure 18, the Western blot analysis result that related protein is expressed behind the demonstration DAM effect BGC-823 cell different time.Figure 18 shows the prolongation of action time along with DAM, and client's albumen (being abbreviated as Akt) of Hsp90 significantly degraded occurs, and the expression of Hsp70 raises, and promptly DAM has Degradation to the Hsp90 client protein.The positive contrast of GA among Figure 18 (geldanamycin).
Figure 19 is the inhibitory action of DAM to human breast cancer cell MDA-MB-231 cell migration.In Figure 19, show that the DAM with 1 μ mol/L and 5 μ mol/L acts on the MDA-MB-231 cell, the migration of its cell and comparing; Obviously be suppressed; Along with the prolongation of action time, DAM is obvious more to the inhibitory action of tumor cell migration, shows that the distance of line hecatomeral cells is far away more.
The specific embodiment
Further set forth the present invention below in conjunction with specific embodiment.
Employing mtt assay detection deacetylase fungal epoxy ethyl ester (concrete steps are following for the strong rhzomorph in south, cytotoxicity DAM):
With RAW264.7 cell (8 * 10
5Individual cells/well) inserts 96 well culture plate overnight incubation; Add bacteria lipopolysaccharide (lipopolysaccharide; LPS) (100ng/ml) and the DAM of variable concentrations (3,6,9,12 μ mol/L; With DMSO as blank) DAM of combined effect 24h or variable concentrations (3,6,9,12 μ mol/L, with DMSO as blank) acts on 24h separately, adds MTT then to final concentration 0.5mg/ml effect 3h; Press and measure OD after 100 μ l/ holes adding lysates (10%SDS, 0.01mol/L HCL) spend the night
590
OD
590The growing state that can reflect cell is therefore through comparing OD
590Value judges whether DAM exists cytotoxicity.
The mtt assay testing result shows that (referring to Fig. 1, Fig. 2): DAM does not have cytotoxicity to the RAW264.7 cell probably under 0～12 μ mol/L concentration conditions.
Through the concentration change of the detection cell proinflammatory factor, thereby distinguish whether deacetylase fungal epoxy ethyl ester (DAM) can suppress the reaction of RAW264.7 cellular inflammation.Concrete test method is following:
1) the DAM Concentraton gradient is set.With RAW264.7 cell (4 * 10
5Cell/mL) inserts 12 well culture plates; Change liquid behind the 24h once; Adding LPS behind 1～2h is that 100ng/ml stimulates 6h to final concentration, adds the DAM (3,6,9,12 μ mol/L, with DMSO as blank) of variable concentrations simultaneously; Observe DAM to the influence that LPS stimulates the proinflammatory factor expression, extract cell culture supernatant and do enzyme linked immunological absorption detection.Through the ELISA detection kit (available from eBioscience company; Mouse TNF-α (Cat#:88-7324-88; Lot#:E09483-1334) and Mouse IL-6 (Cat#:88-7064-88; Lot#:E09362-1631) test kit is each one) detect the concentration of 2 kinds of proinflammatory factors: tumor necrosis factor-alpha in the cell culture supernatant (TNF-α) and interleukin-6 (IL-6).
The ELISA of eBioscience company detection kit detection method is listed below at present.
(1) encapsulates immobile phase antibody.Use Corning Costar 9018ELISA ELISA Plate, shake up adding 100 μ L/ holes, back, ELISA Plate is placed 4 ℃ of reaction overnight with encapsulating the dilution proportion insolubilized antibody of buffer, mixing with 1/250.
(2) clean.Discard solution in the hole, clean 5 times with cleaning buffer solution, each 1min at interval, each every hole washing liquid is no less than 250 μ L, in absorbent paper, beats gently to increase cleaning performance after discarding washing liquid.
(3) sealing.The experiment diluted of 5X to 1X, is added ELISA Plate, room temperature standing and reacting 1h by 200 μ L/ holes.
(4) clean.Same step (2), repeated washing at least 5 times.
(5) add suitable antigen.Use 1X experiment diluent, dilution positive criteria article, positive criteria article 100 μ L/ holes, each concentration do two parallel with guarantee its accurately with reliably.Add the good antigen samples (100 μ L/ hole) of dilution to corresponding hole, cover lid places room temperature standing and reacting 2h or places 4 ℃ to spend the night so that make antigen-antibody reach the maximum combined degree.
(6) clean.Same step (2), repeated washing at least 5 times.
(7) add detection antibody.Use 1X experiment diluent detects antibody with 1/250 dilution proportion, mixes shaking up adding 100 μ L/ holes, back, places room temperature standing and reacting 1h.
(8) clean.Same step (2), repeated washing at least 5 times.
(9) add enzyme labelled antibody.Use 1X experiment diluent with 1/250 dilution proportion enzyme labelled antibody, mix shaking up the back and add 100 μ L/ holes, place room temperature standing and reacting 30min.
(10) clean.Same step (2), in this cleaning step, at interval 1～2min cleans once, and repeated washing at least 7 times.
(11) add reaction substrate.Every hole adds substrate solution 100 μ L, in room temperature standing and reacting 15min.
(12) every hole adds stop buffer 50 μ L, final volume 150 μ L, cessation reaction.
(13) place ELIASA 450nm place to measure its maximum light absorption value (OD) ELISA Plate, and data analysis.
2) LPS Concentraton gradient.With RAW264.7 cell (4 * 10
5Cell/mL) inserts 12 well culture plates; Change liquid behind the 24h once; Add behind 1～2h LPS to final concentration be 100,1000,10000ng/ml stimulates 6h; Add the DAM that final concentration is 12 μ mol/L simultaneously, observe DAM, extract cell culture supernatant and do enzyme linked immunological absorption detection the influence that the LPS of variable concentrations stimulates the proinflammatory factor expression.Through the ELISA detection kit (available from eBioscience company; Mouse TNF-α (Cat#:88-7324-88; Lot#:E09483-1334) and Mouse IL-6 (Cat#:88-7064-88; Lot#:E09362-1631) test kit is each one) detect the concentration of 2 kinds of proinflammatory factors: tumor necrosis factor-alpha in the cell culture supernatant (TNF-α) and interleukin-6 (IL-6).
3) with RAW264.7 cell (8 * 10
5Cell/mL) inserts 96 well culture plate overnight incubation; DAM (3,6,9, the 12 μ mol/L that add LPS (100ng/ml) and variable concentrations then; With DMSO as blank) stimulate 24h, the collecting cell culture supernatant detects the content of nitric oxide (NO) through the Griess method.
Experimental result (referring to Fig. 3～14) shows: DAM can suppress the inflammatory reaction of the inductive RAW264.7 cell of bacteria lipopolysaccharide.DAM is used by having widely in the diseases associated with inflammation medicine of short inflammatory factor mediation at preparation treatment rheumatic arthritis, inflammatory bowel, neurodegenerative disease and septic shock etc.
In this embodiment, adopt Western Blot method to detect the Degradation of deacetylase fungal epoxy ethyl ester (DAM) to the client protein of Hsp90 in the tumor cell.
Use the deacetylase fungal epoxy ethyl ester (DAM) of 1,2.5 and 5 μ g/mL to handle HeLa Cells, human breast carcinoma MCF-7 cell and people's intestinal cancer BGC-823 cell 24h respectively, the GA of 0.5 μ mol/L is as positive control, and equivalent DMSO is as negative control.Culture fluid is removed in suction, with the PBS rinsing once, adds cell pyrolysis liquid, about 4 ℃ of cracking 5min, scrapes fast and gets cell and place the 1.5mL centrifuge tube.Ice-bath ultrasonic cell lysis 10 times, each 1s, the cell pyrolysis liquid that obtains be in 4 ℃, the centrifugal 15min of 13500rpm.Get supernatant, the Bradford method is measured protein concentration, and the adjustment protein concentration is consistent, adds 2 * sample buffer (sample buffer) mixing, and 100 ℃ of water-bath 10min carry out the SDS-PAGE electrophoresis.The 100V electrophoresis changes film 1h to celluloid (PVDF) film that soaked into methanol in advance again.5%BSA room temperature sealing 1h, one anti-(volume ratio dilution in 1: 1000) incubated at room 2h, TBST washing 3 times, each 10min.Two anti-(volume ratio dilution in 1: 5000) incubated at room 1h, the pvdf membrane after the TBST washing develops and observes with enhanced chemical luminous agent (ECL) solution impregnation.
After the deacetylase fungal epoxy ethyl ester of variable concentrations (DAM) is handled tumor cell 24h; Western blot detects the expression with the Hsp90 GAP-associated protein GAP; The result finds; Degraded appears in Akt, the horizontal down-regulation of p-Akt, and degraded appears in relevant protein kinase such as the IKK of nuclear factor signal transduction pathway; The Hsp70 expression raises (Fig. 6,9,10) simultaneously, and is consistent with the exercising result of positive control GA, shows that deacetylase fungal epoxy ethyl ester (DAM) on cellular level, has stronger inhibitory action to Hsp90.
The promptly southern strong rhzomorph of embodiment 4, deacetylase fungal epoxy ethyl ester (DAM) is to the inhibitory action of the activated nuclear factor of inflammatory factor TNF-α (NF-kappaB) signal transduction pathway
In this embodiment; Adopt Western blotting (Western Blot) method and immunofluorescence staining to detect the strong rhzomorph of deacetylase fungal epoxy ethyl ester (DAM) or south among the tumor cell HeLa, the inhibitory action of the activated nuclear factor of inflammatory factor TNF-α (20ng/mL) (NF-kappaB or RelA) signal transduction pathway respectively.
Western Blot method detects the strong rhzomorph in south among the tumor cell HeLa, the inhibitory action of the activated nuclear factor of inflammatory factor TNF-α (NF-kappaB) signal transduction pathway.Use the strong rhzomorph (DAM) in south of 1,2.5 and 5 μ g/mL to handle HeLa cell 23.5h respectively, and activate 0.5h, collect and cell lysis, with nuclear extraction agent extracting nucleus with inflammatory factor TNF-α (20ng/mL).Nuclear factor signaling pathway protein matter in pair cell slurry and the nucleus such as the nuclear situation of going into of nuclear factor (RelA) and CKIs matter I κ B thereof detect respectively, and the result finds that the nuclear effect of going into of nuclear factor (RelA) is suppressed (Fig. 7).
Immunofluorescence staining detects the inhibitory action of the promptly southern strong rhzomorph of deacetylase fungal epoxy ethyl ester (DAM) to the activated nuclear factor of inflammatory factor TNF-α (NF-kappaB) signal transduction pathway.Handle HeLa cell 23.5h with the strong rhzomorph (DAM) in the south of 2.5 μ g/mL, activate 0.5h with inflammatory factor TNF-α (20ng/mL) again, collecting cell carries out nuclear staining with DAPI, and nuclear factor RelA (p65) is dyeed through immunofluorescence staining.The result finds that nuclear factor RelA goes into the nuclear effect clearly under inflammatory factor TNF-α (20ng/mL) activation, and nucleus occurs significantly orange.Through with nuclear staining after blue superimposed, the nucleus that has RelA to go into the nuclear effect presents pink; And in the HeLa cell with strong rhzomorph (DAM) processing in south, though activate through inflammatory factor TNF-α (20ng/mL), the nuclear of going into of nuclear factor is prevented from, and whether no matter whole nucleus superpose all presents blueness (Fig. 8).
Nuclear factor is gone into the nuclear back through combining to start relevant cell survival genetic transcription with DNA, thereby makes inflammatory factor expression such as cytokine, cell survival.And the strong rhzomorph (DAM) in south is gone into nuclear through stoping nuclear factor, and reaches effect such as corresponding antiinflammatory.
The promptly southern strong rhzomorph of embodiment 5, deacetylase fungal epoxy ethyl ester (DAM) is to the inhibitory action of human breast cancer cell MDA-MB-231 migration
All apparatuses that need sterilize are used preceding sterilization, and ruler and marker pen be ultra-vioket radiation 30min (in the super-clean bench) before operation.Culture medium: the DMEM serum-free medium, cultivate with 24Well ComboPlate
1, earlier at 24 orifice plates behind, comparing, evenly drawing horizontal line, approximately whenever at a distance from 0.5～1cm together, crossing via hole with ruler with the marker pen.
2, shop dish density: 1.0 * 105/ml, dosing behind the dish 24h of shop.
3, comparing ruler with the rifle head, hanging down as for horizontal line cut behind as far as possible, it is vertical that the rifle head is wanted, and can not tilt.
4, wash cell 3 times with PBS, remove free cell, add serum-free medium.
5, put into 37 ℃ of degree 5%CO
2Incubator is cultivated.Take a sample with 48h by 0,24,36, take pictures.
Claims (4)
1. deacetylase fungal epoxy ethyl ester is in the application of preparation Hsp90 inhibitor, and the chemical formula of said deacetylase fungal epoxy ethyl ester is C
14H
16O
4, molecular weight is 246, its chemistry 2-(8-methyl-9-oxa--dicyclo [4.2.1] nine-2,4-diene-7-yl) by name-6-oxygen-3, and 6-dihydro-2H-pyrone is white pinniform or bar-shaped transparent crystallization, chemical structural formula is:
2. deacetylase fungal epoxy ethyl ester is in the application of preparation Hsp90 inflammation inhibitor, and the chemical formula of said deacetylase fungal epoxy ethyl ester is C
14H
16O
4, molecular weight is 246, its chemistry 2-(8-methyl-9-oxa--dicyclo [4.2.1] nine-2,4-diene-7-yl) by name-6-oxygen-3, and 6-dihydro-2H-pyrone is white pinniform or bar-shaped transparent crystallization, chemical structural formula is:
3. the application of deacetylase fungal epoxy ethyl ester in the preparation treatment diseases associated with inflammation medicine alpha mediated by inflammatory factor TNF-, the chemical formula of said deacetylase fungal epoxy ethyl ester is C
14H
16O
4, molecular weight is 246, its chemistry 2-(8-methyl-9-oxa--dicyclo [4.2.1] nine-2,4-diene-7-yl) by name-6-oxygen-3, and 6-dihydro-2H-pyrone is white pinniform or bar-shaped transparent crystallization, chemical structural formula is:
4. like the said application of claim 3, it is characterized in that said diseases associated with inflammation comprises rheumatic arthritis, inflammatory bowel or neurodegenerative disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110201969 CN102319240A (en) | 2011-07-19 | 2011-07-19 | Application of deacetylmycoepoxydiene serving as Hsp90 inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110201969 CN102319240A (en) | 2011-07-19 | 2011-07-19 | Application of deacetylmycoepoxydiene serving as Hsp90 inhibitor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102319240A true CN102319240A (en) | 2012-01-18 |
Family
ID=45447168
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110201969 Pending CN102319240A (en) | 2011-07-19 | 2011-07-19 | Application of deacetylmycoepoxydiene serving as Hsp90 inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102319240A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749306A (en) * | 2016-11-30 | 2017-05-31 | 厦门大学 | DAM and its derivative as marine antifoulant application |
| CN113521314A (en) * | 2014-09-17 | 2021-10-22 | 纪念斯隆-凯特琳癌症中心 | HSP 90-Targeted inflammation and infection imaging and treatment |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101590036A (en) * | 2009-06-26 | 2009-12-02 | 厦门大学 | Epoxy diene is as the application of short inflammatory mediator inhibitor |
-
2011
- 2011-07-19 CN CN 201110201969 patent/CN102319240A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101590036A (en) * | 2009-06-26 | 2009-12-02 | 厦门大学 | Epoxy diene is as the application of short inflammatory mediator inhibitor |
Non-Patent Citations (2)
| Title |
|---|
| 《厦门大学学报(自然科学版)》 20091130 周显凤等 反相柱层析制备去乙酰真菌环氧乙酯 参见第871-874页 1-4 第48卷, 第6期 * |
| 《厦门大学硕士学位论文》 20091215 徐莹 红树真菌的生物活性以及去乙酰真菌环氧乙酯的发酵和理化性质的研究 参见第11页第2段 1 , * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113521314A (en) * | 2014-09-17 | 2021-10-22 | 纪念斯隆-凯特琳癌症中心 | HSP 90-Targeted inflammation and infection imaging and treatment |
| CN106749306A (en) * | 2016-11-30 | 2017-05-31 | 厦门大学 | DAM and its derivative as marine antifoulant application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11117919B2 (en) | Icariside compound, preparation method thereof, and application thereof | |
| Raja et al. | Recent developments in therapeutic applications of Cyanobacteria | |
| Carbone et al. | Evaluation of microalgae antiviral activity and their bioactive compounds | |
| US8313779B2 (en) | Evaluation method and screening method for substance having action of activating/suppressing innate immunity, agent and food product for activating/suppressing innate immune mechanism and method for producing the same | |
| Li et al. | Activation of RAW 264.7 cells by a polysaccharide isolated from Antarctic bacterium Pseudoaltermonas sp. S-5 | |
| Ibrahim et al. | New cytotoxic cyclic peptide from the marine sponge-associated Nocardiopsis sp. UR67 | |
| CN105085711B (en) | A kind of preparation method and applications of chitosan oligosaccharide | |
| Guan et al. | Adjuvant effects of salidroside from Rhodiola rosea L. on the immune responses to ovalbumin in mice | |
| Aminin et al. | Immunomodulatory action of monosulfated triterpene glycosides from the sea cucumber Cucumaria okhotensis: Stimulation of activity of mouse peritoneal macrophages | |
| Lee et al. | (10 Z)-Debromohymenialdisine from Marine Sponge Stylissa sp. Regulates Intestinal Inflammatory Responses in Co-Culture Model of Epithelial Caco-2 Cells and THP-1 Macrophage Cells | |
| CN106543271A (en) | Anti-drug resistance infection peptide C bf 14 2 and application thereof | |
| Kim et al. | Immunomodulatory activity of extracellular vesicles of kimchi-derived lactic acid bacteria (Leuconostoc mesenteroides, Latilactobacillus curvatus, and Lactiplantibacillus plantarum) | |
| CN105732560B (en) | Siskin isoflavonoid derivative and preparation method thereof and the application in pharmacy | |
| CN100391940C (en) | Compounds | |
| CN102319240A (en) | Application of deacetylmycoepoxydiene serving as Hsp90 inhibitor | |
| Rubanova et al. | Pseurotin D inhibits the activation of human lymphocytes | |
| CN102212581A (en) | Preparation method and application of cordyceps polysaccharide germanium | |
| CN101724631A (en) | Preparation of functional area of sea purse blood vessel growth inhibition factor 1 and use of the functional area of sea purse blood vessel growth inhibition factor 1 in medicaments for preventing and curing tumors | |
| Tan et al. | A novel CXCR4 targeting protein SDF-1/54 as an HIV-1 entry inhibitor | |
| Liu et al. | Immunomodulatory activity of butanol extract from Solanum lyratum in tumor-bearing mice | |
| CN110327328A (en) | Application of 25 β--23 β of the secondary cyclobutenyl-isobutyl acyloxy mibemycin derivative in antitumor | |
| Kotajima et al. | Axl, immune checkpoint molecules and HIF inhibitors from the culture broth of Lepista luscina | |
| CN102424802B (en) | Bacillus pumilus, strain culture method, and application thereof | |
| CN103739690A (en) | Method for separation preparation of anti-tumor polypeptide compound from ArcainflataReeve, and uses of anti-tumor polypeptide compound | |
| JP2009209132A (en) | Antiallergic composition using new strain, lactobacillus crispatus kt-11, kt-23 and kt-25 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120118 |