CN109481425A - Application of the agrimophol B as TFEB nuclear translocation inducer - Google Patents
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Abstract
The present invention relates to the applications of agrimophol B, specifically, present invention firstly discovers that agrimophol B can induce TFEB nuclear translocation, i.e. induction TFEB from Chromosome migration to nucleus in, the effect of its transcription factor can be played, the variation of downstream passages is caused in conjunction with gene.Present invention demonstrates that agrimophol B can induce cell autophagy.It is of the invention the result shows that, agrimophol B can reduce the accumulation of intracellular cholesteryl and phosphatide, so as to be accumulated as treatment cholesterol and phosphatide caused by related disease potential drug.
Description
Technical field
The present invention relates to agrimophol B, and in particular to agrimophol B is answered as the inducer for inducing TFEB nuclear translocation
With, and treating the application in accumulation of cholesterol relevant to TFEB nuclear translocation and phosphatide accumulation disease.
Background technique
Adjusting cellular cholesterol levels is essential for the function of normal cell and development.One intracellular gallbladder
Partly transport by cholesterol between various compartments and film is adjusted sterol levels.Cholesterol between various cell membranes
Suitable distribution is particularly significant for many biological functions, as signal transduction is exchanged with film.Cholesterol levels can also pass through fortune
It is defeated to be reconciled from cell to the extracellular receptor of Cholesterol removal.These cholesterol transport mechanism have been widely studied, and
Adjust cellular cholesterol levels defect oneself link together with various diseases.
Niemann-Pick disease is a kind of genetic, disease related with lysosome storage obstacle.Currently, Niemann-Pick disease has
The therapeutic scheme of effect is very limited, no special treatment.There are four types of types for Niemann-Pick disease: A, B, C and D type.C-type Niemann-Pick disease
(NPC) be a kind of recessive hereditary disease, can cause cholesterol and other lipids in the cell of many types not
Normal accumulation.
There are two the mankind, and gene is associated with NPC defect, NPC1 and NPC2.NCP1 is the memebrane protein of a multi-span, is led to
Often combine with the endosome in advanced stage or lysosome, the cholesteryl ester of cell is brought in organelle of degrading, hydrolysis into through lipoprotein.NPC1 is lacked
It falls into and may cause cell autophagy decrease and accumulation of cholesterol, and then cause serious nerve degeneration and liver dysfunction, that is, develop
As Niemann-Pick disease (Dorothea Maetzel, Sovan Sarkar, Haoyi Wang, et al.Genetic and
Chemical Correction of Cholesterol Accumulation and Impaired Autophagy in
Hepatic and Neural Cells Derived from Niemann-Pick Type C Patient-Specific
iPS Cells,Stem Cell Reports,2014,2,866-880).Since NPC1 protein function lacks, lead to cholesterol generation
Thank to exception.There is cholesterol metabolism abnormity caused by studies have shown that NPC1 afunction related with autophagy dysregulation in the recent period.
Phospholipidosis is that a phosphatide has the case where excessive accumulation in bodily tissue.The excessive accumulation of phosphatide is considered as
It is related with the change of the synthesis of phosphatide and/or metabolism.When patient is given with certain drug, phospholipidosis can occur.For example, working as
Phospholipidosis can be caused when to mankind's amiodarone, perhexiline, Prozac, gentamicin.
Therefore, disease and Phospholipids caused by defect are adjusted as cellular cholesterol levels for Niemann-Pick disease etc.
Disease, it is also necessary to study new effective treatment means.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of TFEB nuclear translocation inducer agrimophol B, i.e. induction TFEB is from thin
Cytoplasm is transferred in nucleus, and thereby reduces the accumulation of intracellular cholesteryl and phosphatide.
An aspect of of the present present invention provides application of the agrimophol B as TFEB nuclear translocation inducer, wherein the red crowned crane
The chemical structural formula of careless phenol B is as described below
Further, above-mentioned agrimophol B is clinical treatment as the application of TFEB nuclear translocation inducer.
Further, clinical treatment is treatment accumulation of cholesterol related disease.
Further, above-mentioned clinical treatment is treatment phosphatide accumulation related disease.
Further, above-mentioned phosphatide accumulation related disease is phospholipidosis.
Another aspect of the present invention provides agrimophol B in preparation for treating by inducing TFEB nuclear translocation to be treated
Disease drug in application, the chemical structural formula of agrimophol B is as described below
Further, above-mentioned disease is accumulation of cholesterol related disease.
Further, above-mentioned disease is phosphatide accumulation related disease.
Further, above-mentioned phosphatide accumulation related disease is phospholipidosis.
Another aspect of the present invention provides application of the agrimophol B as the inducer of cell autophagy, the change of agrimophol B
It is as described below to learn structural formula
Definition: nuclear translocation, nuclear translocation of the invention refer to that intracytoplasmic substance enters nucleus.
Hairyvein agrimony and agrimophol B, the medicinal source of hairyvein agrimony are rosaceous plant Agrimony Agrimonia pilosa
Ledeb, hairyvein agrimony are mainly produced in the ground such as Zhejiang, Jiangsu, Hubei.In traditional clinical application, there is hairyvein agrimony convergence to stop
The effect of blood, stop dysentery, preventing malaria, qi-restoratives, is conventionally used to treat out blood trouble, diarrhea, dysentery, malaria fever and chills, de-power internal lesion caused by overexertion.Hairyvein agrimony
Phenol B is derived from a polyphenol compound for Chinese medicine hairyvein agrimony, is phloroglucin structure.
Cholesterol, also known as cholesterine.A kind of derivative of cyclopentanoperhy drophenanthrene.Cholesterol is widely present in animal body,
Especially with the abundantest in brain and nerve fiber, content is also high in kidney, spleen, skin, liver and bile.Its dissolubility and fats
Seemingly, not soluble in water, it is soluble in ether, chloroform equal solvent.Cholesterol is the indispensable important substance of institute, animal tissue cell, it
It is not only involved in form cell membrane, and is synthetic bile acid, the raw material of vitamin D and steroid hormone.
TFEB (transcription factor EB, transcription factor EB), be leucine zipper b HLH-Zip transcription because
Member in sub-family in Mi T/TFE family.There is article report, TFEB participates in the adjusting of lysosome synthesis and function, and regulation is thin
Born of the same parents' autophagy, and it is related to a variety of diseases such as neurodegenerative disease, cancer and lysosomal storage disease.Recently it has been reported that, mistake
The dopamine neuron apoptosis that expression TFEB can prevent MPTP from inducing；The activation energy of TFEB mitigates alpha-synapse nucleoprotein in brain group
The damage of nerve cell caused by the abnormal aggregation and oxidative stress and inflammation knitted.
LC3 (Light Chain 3, light chain 3), is the labelled protein on autophagosome film.It is intracellular there are two kinds of forms
LC3 albumen, LC3- I and LC3- II.Intracellular its C-terminal of newly synthesized LC3 becomes cytoplasm soluble form by Atg4 proteolytic cleavage
LC3- I.After autophagosome is formed, LC3- I is clipped and ubiquitination processes the phosphatidyl ethanol for modifying and autophagosome film surface
Amine (PE) coupling, becomes the LC3- II of film combining form and is positioned at autophagosome inner membrance and outer membrane.It is positioned at certainly with some other
The Atg albumen bitten on body film is different (only playing a role in a certain stage of autophagy process), is retained in II all-the-time stable of LC3-
On autophagosome film until with lysosome fusion, therefore be used as the mark molecule of autophagosome.The content or LC3- of LC3- II
The ratio of II/LC3- I and the quantity of autophagosome are positively correlated, and reflect the autophagy activity of cell to a certain extent.
Beneficial effects of the present invention:
1, present invention firstly discovers that agrimophol B can induce TFEB nuclear translocation, i.e. induction TFEB from Chromosome migration to
In nucleus, to play transcription factor effect in conjunction with gene, cause a series of variations in downstream.
2, present invention demonstrates that agrimophol B can induce cell autophagy.Cell autophagy is that lysosome degrades to cellular content
Process, be the example of most effective cellular content cycling and reutilization, autophagy has the function of regulating cell energy, facilitates
Keep the positive balance of cellular energy.Effect of the autophagy in cell metabolism is always its major function.Cell autophagy is in addition to decomposing
Albumen supplements intracellular amino acid, facilitate lipid within endothelial cells, glycogen mobilization and hydrolysis other than, also by mitochondria from
The form bitten controls the quantity and function of mitochondria network, the final metabolism for realizing cell, energetic supersession, thin
Born of the same parents update.
3, of the invention the results show that agrimophol B can reduce the accumulation of intracellular cholesteryl and phosphatide, so as to
The potential drug of related disease caused by being accumulated as treatment cholesterol and phosphatide.
Detailed description of the invention
Fig. 1 is agrimophol B chemical structure；
TFEB protein fluorescence image in U2OS-TFEB cell after Fig. 2A is various concentration agrimophol B processing 4 hours；
Fig. 2 B shows that agrimophol B induces TFEB nuclear translocation efficiency；
Fig. 2 C shows that agrimophol B induces TFEB nuclear translocation nuclear area TFEB fluorescence intensity；
Fig. 3 A is GFP-LC3 and mCherry-LC3 fluorescence imaging figure；
Fig. 3 B shows that agrimophol B handles the concentration effect relationship formed to GFP-LC3；
Fig. 3 C shows that agrimophol B handles the concentration effect relationship formed to mCherry-LC3；
Fig. 3 D shows that agrimophol B forms function analysis to GFP-mCherry-LC3 albumen；
Fig. 3 E shows that agrimophol B acts on the influence to U2OS-mCherry-Green-LC3 cell quantity in 4 hours；
Fig. 4 A is cholesterol Filipin III mark fluorescent imaging in GM03123 cell after agrimophol B is handled 48 hours
Figure；
Fig. 4 B is that CMRA marks GM03123 cytoplasmic fluorescence image after agrimophol B is handled 48 hours；
Fig. 4 C shows that agrimophol B acts on counting to cholesterol in 48 hours and analyzes；
Fig. 4 D shows that agrimophol B acts on 48 hours to Filipin III Fluorescence Intensity Assays；
Fig. 4 E shows that agrimophol B handles the influence to GM03123 cell quantity in 48 hours；
Fig. 5 A is that GM03123 cellular cholesterol Filipin III mark fluorescent is imaged after agrimophol B is handled 72 hours；
Fig. 5 B is that CMRA marks the imaging of GM03123 cytoplasmic fluorescence after agrimophol B is handled 72 hours；
Fig. 5 C shows that agrimophol B acts on counting to cholesterol in 72 hours and analyzes；
Fig. 5 D shows that agrimophol B acts on 72 hours to Filipin III Fluorescence Intensity Assays；
Fig. 5 E shows that agrimophol B handles the influence to GM03123 cell quantity in 72 hours；
Fig. 6 A is HepG2 cellular phospholipid and nucleus fluorescence imaging figure after agrimophol B processing；
Fig. 6 B shows agrimophol B concentration and HepG2 cellular phospholipid content concn effect relation；
Fig. 6 C shows the concentration effect relationship of phosphatide points and agrimophol B；
Fig. 6 D shows the concentration effect relationship of HepG2 cell quantity and agrimophol B.
Specific embodiment
Below with reference to embodiment, in order to explain the technical scheme of the invention in detail.Reagent used in embodiment, is set instrument
Standby can disclose obtains.
Laboratory apparatus used in this experiment mainly has: ImageExpress high intension phosphorimager；Cellomics
ArrayScan high intension fluoroscopic imaging systems.
Laboratory sample: agrimophol B
Embodiment 1: compound is prepared
(1) it prepares agrimophol B: weighing agrimophol B powder and be dissolved in DMSO and be made into 10mmol/L storing liquid, be stored in-
20℃.In vitro cell experiment uses after being diluted with DMEM complete medium.
(2) prepare rapamycin (rapamycin): weigh rapamycin powder be dissolved in DMSO be configured to 10mmol/L storage
Liquid is stored in -20 DEG C.In vitro cell experiment is diluted to 1 μM of use with DMEM complete medium.
(3) it prepares Torin 1: weighing 1 powder of Torin and be configured to 1mmol/L storing liquid, be stored in -20 DEG C.It is external real
It tests and is diluted to 250nM use with DMEM complete medium.
(4) it configures SAHA solution: weighing SAHA powder and be configured to 100mmol/L storing liquid, be stored in -20 DEG C.It is external real
It tests and is used with after the dilution of DMEM complete medium.
Embodiment 2: cell culture
U2OS-TFEB-EGFP cell culture is in DMEM complete medium, until when growth conditions are good, with 11,000/hole
Cell number is inoculated in overnight incubation on the black wall dianegative in 96 holes.Compound to be tested is added in next day, detects TFEB after being incubated for 4h
Nuclear translocation effect.
U2OS-mCherry-EGFP-LC3 stable expression cell line is incubated at containing 2 μ g/mL puromycin culture mediums, until
Growth conditions are good, complete using the DMEM without puromycin with 6500/hole cell inoculation on the black wall dianegative in 96 holes
Full culture medium overnight incubation.Next day after being added untested compound four hours, detects LC3 expression quantity.
GM03123 cell is used using the EMEM culture medium culture for containing 10% fetal calf serum.To logarithmic growth phase, by cell
It is inoculated on the black wall dianegative in 96 holes with 2400/hole, overnight incubation.Evaluation compound to be measured is added in next day, cultivates respectively
48 hours and 72 hours progress fluorescent markers.
HepG2 is incubated at DMEM complete medium, contains 10% fetal calf serum, 100U/mL streptomycin sulphate and 100 μ g/mL
Penicillin.Fresh culture is replaced in recovery second day.Progress cell passage in every 2-3 days, well carries out to cell growth state
Pharmaceutical activity research.Passage number amount is within 14 generations.
Embodiment 3: agrimophol B induces TFEB nuclear translocation Effect study
It is respectively 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μ that agrimophol B, which is diluted to concentration,
M, 20 μ L are drawn respectively be added in the U2OS-TFEB-EGFP cell containing 180 μ L culture mediums for 1.5625 μM, so that final concentration
Respectively 20 μM to 156.25nM.In parallel laboratory test, positive control is used as using 1 μM of rapamycin and 250nM Torin 1.
Using not medicine treatment group cell as negative control.Meanwhile being respectively adopted without glucose, be free of glucose and glutamy
Control of the culture medium of amine as nutrition starvation.After drug incubation handles 4h, using 4% paraformaldehyde, the cells are fixed.Using
10 μ g/mL Hoechst 33342 mark nucleus, take 100 μ L fluorescent dyes, are incubated for jointly with cell 15 minutes.Moistened with PBS
It washes 3 times, acquires TFEB and nucleus fluorescence picture with MD ImageXpress.As shown in Fig. 2, agrimophol B can induce TFEB
Nuclear translocation.The inventors discovered that agrimophol B can significantly activate TFEB to enter core at 10 μM, cell positive rate is close to 100%.
Further study show that promoting to drop rapidly under the action of TFEB nuclear translocation as concentration reduces.The setting for optimizing concentration, by 2
μM successively successively decrease, can agrimophol B induction TFEB nuclear translocation concentration effect variation visible in detail, about there is inflection point at 5 μM,
The not significant inducing cell quantity loss of agrimophol B in evaluated concentration range.The application shows that agrimophol B swashs for the first time
TFEB nuclear translocation effect living has potential adjusting cell metabolism effect.Phospholipidosis is characterized in that phosphatide accumulation enters lyase
Body.Studies have shown that this phosphatide accumulation has activated the lysosome-nuclear signal conduction mediated by TFEB, lyase can be promoted
Body biosynthesis, autophagy and encytosis etc., it is possible to the aversion response as a kind of degradation accumulation phosphatide.Wherein, TFEB
It is autophagy-lysosome system key regulator, and drug-induced phosphatide accumulation can be reduced when TFEB is overexpressed.
This discovery shows that TFEB has protective effect to the accumulation of phosphatide.
Embodiment 4: the cell autophagy stream that agrimophol B induces LC3 to mediate
With DMEM complete medium by agrimophol B be configured to concentration be respectively 100 μM, 30 μM, 10 μM, 3 μM, 1 μM,
0.3 μM of working solution is drawn 20 μ L and is added in the U2OS-mCherry-EGFP-LC3 cell containing 180 μ L culture mediums.It is incubated for
4 as a child, marks nucleus using 10 μ g/mL Hoechst, 33342 solution.After being incubated for 15 minutes, after PBS rinse 3 times,
MCherry-LC3 is acquired with ImageXpress Micro Confocal High-Content Imaging System platform,
GFP-LC3 and cell nuclear information.It is average each intracellular using the analysis of Custom module (custom block) method
The number of mCherry-LC3, GFP-LC3, number and the cell quantity variation of mCherry-EGFP-LC3.Fig. 3 A indicates hairyvein agrimony
LC3 fluorescence imaging figure when phenol B acts on 4h, Vehicle (blank) indicates negative control group, the results show that negative control group cell
There are a certain number of LC3 to be distributed.Torin 1 (250nM) indicates positive controls, is remarkably reinforced after Torin1 (250nM) processing
GFP-LC3 and mCherry-LC3 points.GFP-LC3 and mCherry-LC3 and common location LC3 is remarkably reinforced in agrimophol B
Formation.Fig. 3 B indicates GFP-LC3 fluorescence intensity data analysis chart, and data analysis result shows that agrimophol B dose-dependant promotees
It is formed into GFP-LC3, under 10 μM of effects, the amount that GFP-LC3 is formed is 8 times of negative control or so.(6.67-10 μ when high concentration
M), with extended durations of action, GFP-LC3 accumulation is increased slightly.When low concentration (1.32-4.44 μM), as action time prolongs
Long, GFP-LC3, which is formed, is presented decreasing trend.Fig. 3 C indicates that mCherry-LC3 fluorescence intensity data analysis chart, agrimophol B are made
With rear, mCherry-LC3 expression is dramatically increased, processing 4h obviously than 2h when cumulant it is more.When increasing the drug incubation time to 6h,
MCherry-LC3 expression does not continue to increase, and slightly has reduced trend instead.Show that agrimophol B remarkably promotes autophagy process
Middle early stage autophagosome is formed.Fig. 3 D indicate mCherry-EGFP-LC3 area ratio analyze datagram, with mCherry-LC3 with
The direction of GFP-LC3 area ratio (mCherry/GFP Area Ratio) evaluation cell autophagy stream.Normal group cell is total
LC3 amount is the ratio of cytoplasm LC3 content between 1.6-2.0, shows that cell has certain level autophagy stream.Torin 1 exists
The ratio of different time processing group is suitable with negative control group, maintains the autophagocytosis of cell.Agrimophol B is at high concentration
After managing 2-6h, the level of mCherry/GFP is slightly reduced, but still is higher than 1, illustrates still have a certain amount of autophagy lysosome to be formed.
When concentration is reduced to 4.4 μM, ratio restores to 1.6 or so.Agrimophol B is handled in 4-6h, and LC3 cumulative amount does not become significantly
Change, after showing that action time is more than 4h, cell autophagy corpusculum forms and do not continue to increase.Fig. 3 E indicates agrimophol B to U2OS-
The influence of mCherry-EGFP-LC3 cell quantity.Negative control group cell and the cell quantity of positive controls are without obvious poor
Different, the cell quantity of positive controls is reduced with the increase of action time, after agrimophol B processing, with the drop of concentration
Low, cell quantity is also being reduced, and it is relatively high that wherein the cell quantity of drug-treated 4h, which is compared with processing 2h with 6h,.
Embodiment 5: agrimophol B reduces the sedimentation of GM03123 cellular cholesterol
It is 300 μM, 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM that agrimophol B is diluted to concentration respectively
Working solution, take 20 μ L to be added to the culture medium containing 180 μ L so that final concentration be respectively from 30 μM according to 3 times of gradients successively
Dilution.Respectively after compound incubation 48 hours and 72 hours, 30 minutes are incubated for mark in 37 DEG C of conditions with 5 μM of CMRA
Remember full cell.With the cholesterol in 25 μ g/mL Filipin fluorochrome label cells.With Thermo Scientific HCS
VTI acquires fluorescence information.Cell is identified according to CMRA, identifies cholesterol fluorescent marker with Spot, is analyzed average each intracellular
Points (Spot count/cell) and point intensity (Spot Intensity/cell).Fig. 4 A and 4B indicate to use Fillipin
With GM03123 cellular cholesterol fluorogram after the various concentration agrimophol B processing 48h of CMRA label cell quantity label.Figure
After 5A and 5B indicates the various concentration agrimophol B processing 72h using Fillipin and CMRA label cell quantity label
GM03123 cellular cholesterol fluorogram GM03123 cell is the cell in NPC1 saltant type patient source, thin as accumulation of cholesterol
Born of the same parents' model.GM05659 is the cell in Healthy People source, 0.1%DMSO is added as negative control group, 10 μM of SAHA are the positive
Control group is analyzed after various concentration compound agrimophol B is respectively acting on GM03123 cell 48h and 72h using Filipin
Endocellular liberation cholesterol level and cell quantity.Agrimophol B handles 48h and reaches strongest inhibition when concentration is 10 μM
Rate, handling strongest inhibiting rate when 72h is 3.3 μM.The effect of 30 μM of agrimophol B processing 48h and 72h is weaker than low concentration
Group, the concentration effect having a rebound.U-shaped is presented in the concentration-response curve of two processing time groups, and IC50 is 0.94 μ respectively
M and 0.68 μM.Agrimophol B processing 48h does not show strong cytotoxicity, and cell quantity has the tendency that slightly decreasing.With
Extended durations of action to 72h, there is obvious cell quantity loss (Fig. 3-6) in 10-30 μM of processing group.Table is studied herein
Bright, agrimophol B, which has, significantly improves cholesterol deposition effect, and more excellent condition is 3 μM of processing 72h.
Embodiment 6: agrimophol B inhibits phosphatide sedimentation research
By 96 porocyte culture plates with collagen I (30 holes μ L/, concentration be 100 μ g/mL) in 37 DEG C of incubation 30min,
It uses PBS rinse 3 times again.HepG2 cell is inoculated in 96 orifice plates according to the density in 5,500/hole, every hole is cultivated completely containing DMEM
100 μ L of base.Tissue culture plate is placed in 37 DEG C of cell incubators to be incubated overnight.
Next day prepares phosphatide according to the operating process that kit is recommended and detects fluorescent dye.By LipidTOXTM Red
Phospholipidosis fluorescent reagent DMEM complete medium dilutes 500 times and is prepared into 2 × fluorescent dye, crosses 0.2 μm of filter
Film.With the fluorescent dye dilute induction liquid at 20 μM of amiodarones (Amiodarone) or 60 μM of inderals (propranolol), make
For the inducer that cellular phospholipid is formed, tissue culture plate is added to by 50 holes μ L/.
Meanwhile 2 × compound solution is prepared, it is added in cell according to 50 holes μ L/, makes 100 hole μ L/ of cell liquid volume.
When activity verifying, the concentration of agrimophol B is respectively as follows: 10 μM, 8 μM, 6 μM, 4 μM, 2 μM, 0 μM.
Control group is respectively as follows: vehicle control group (vehicle) inducer containing phosphatide, and blank control group is induced without phosphatide
Agent.All cells are added fluorescent dye and are incubated for jointly.Each compound repeats 3 multiple holes.Tissue culture plate is placed in 37
DEG C cell incubator culture 48h.
Cell is fixed
(1) cell culture medium is discarded, the 4%PFA of 100 μ L preheating is added in every hole, places 20min in room temperature condition, will be thin
Born of the same parents fix.
(2) with PBS by cell rinse 3 times, PBS100 μ L is added every time.
(3) 100 μ L Hoechst, 33342 solution (concentration is 10 μ g/mL) is added in every hole, is incubated in room temperature condition
15min marks nucleus.
(4) with PBS by cell rinse 3 times, PBS100 μ L is added every time.
(5) every hole is used for fluorescence imaging after 100 μ L PBS are added.
Phosphatide fluorescence analysis
Fluorescence signal is acquired in Collomic ArrayScan TVI high intension platform, using 10 × object lens, nucleus letter
Number Ex/Em:386/461nm；Acquisition lipid signaling condition is Alexa Fluor 594/Texas Red filter.Every hole acquires 6
The visual field.
Using the every hole cell number of the analytical of Spot Detection (Cell count), average each cell phosphorus
The fluorescence intensity (Spot Intensity/Cell) of rouge number (Spot count/cell) and phosphatide.Fig. 6 indicates agrimophol B
To the inhibiting effect that the phosphatide of Propranolol induction is formed, Fig. 6 A indicates HepG2 intracellular phospholipid and nucleus fluorescence picture, knot
Fruit shows that agrimophol B significantly reduces intracellular content of phospholipid, significantly reduces cell quantity.It is right at 4-10 μM
HepG2 cells show goes out more stable inhibiting effect, and the inhibiting rate to cell is about 75%, cell number and negative control at 2 μM
Group is relatively.Fig. 6 B, 6C, 6D indicate HepG2 cell quantity, phosphatide drop quantity and fluorescence intensity after agrimophol B processing 48h
Concentration-response curve show good concentration dependant effect relation in 10 μM, it is raw that phosphatide can be completely inhibited in 4-10 μM
There is a part phosphatide drop at, 2 μM of whens, but to lack fluorescence weaker for expression.Although cell quantity is reduced, the content of phosphatide
Still there is certain space between cell quantity.The result of study further confirms that agrimophol B can inhibit drug-induced phosphorus
Rouge nucleus formation.
Above-mentioned technical proposal only embodies the optimal technical scheme of technical solution of the present invention, those skilled in the art
The principle of the present invention is embodied to some variations that some of them part may be made, belongs to the scope of protection of the present invention it
It is interior.
Claims (10)
1. application of the agrimophol B as TFEB nuclear translocation inducer, wherein the chemical structural formula of the agrimophol B is as follows
It is described
2. application according to claim 1, which is characterized in that the application is used for clinical treatment.
3. application according to claim 2, which is characterized in that the clinical treatment is treatment accumulation of cholesterol correlation disease
Disease.
4. application according to claim 2, which is characterized in that the clinical treatment is that treatment phosphatide accumulates related disease.
5. application according to claim 4, which is characterized in that the phosphatide accumulation related disease is phospholipidosis.
6. agrimophol B is used to treat the application in the drug for being able to the disease treated by induction TFEB nuclear translocation in preparation,
Wherein, the chemical structural formula of agrimophol B is as described below
7. application according to claim 6, which is characterized in that the disease is accumulation of cholesterol related disease.
8. application according to claim 6, which is characterized in that the disease is that phosphatide accumulates related disease.
9. application according to claim 8, which is characterized in that the phosphatide accumulation related disease is phospholipidosis.
10. application of the agrimophol B as the inducer of cell autophagy, wherein the chemical structural formula of agrimophol B is as described below
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110724203A (en) * | 2019-11-08 | 2020-01-24 | 中国人民解放军第四军医大学 | Short peptide for promoting TFEB (T-Epstein-Barr) nuclear translocation, linear short peptide based on short peptide and application of short peptide in relieving cerebral ischemic injury |
| CN114209742A (en) * | 2021-12-27 | 2022-03-22 | 图方便（苏州）环保科技有限公司 | Clostridium perfringens inhibitor and application thereof |
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| CN110724203A (en) * | 2019-11-08 | 2020-01-24 | 中国人民解放军第四军医大学 | Short peptide for promoting TFEB (T-Epstein-Barr) nuclear translocation, linear short peptide based on short peptide and application of short peptide in relieving cerebral ischemic injury |
| CN114209742A (en) * | 2021-12-27 | 2022-03-22 | 图方便（苏州）环保科技有限公司 | Clostridium perfringens inhibitor and application thereof |
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