CN109481425B

CN109481425B - Application of agrimonol B as TFEB nuclear translocation inducer

Info

Publication number: CN109481425B
Application number: CN201811548849.7A
Authority: CN
Inventors: 裴艳玲; 郭毅; 李凡磊
Original assignee: Hebei Xinminhe Medicine Technology Development Co ltd
Current assignee: Hebei Xinminhe Medicine Technology Development Co ltd
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2021-06-08
Anticipated expiration: 2038-12-18
Also published as: CN109481425A

Abstract

The invention relates to application of agrimophol B, and particularly discloses that agrimophol B can induce TFEB nuclear translocation, namely TFEB is induced to be transferred from cytoplasm to nucleus, so that the TFEB can be combined with gene to play a role of transcription factor to cause change of a downstream pathway for the first time. The invention proves that agrimonol B can induce autophagy of cells. The results of the invention show that the agrimophol B can reduce the accumulation of cholesterol and phospholipid in cells, thereby being used as a potential drug for treating related diseases caused by the accumulation of cholesterol and phospholipid.

Description

Application of agrimonol B as TFEB nuclear translocation inducer

Technical Field

The invention relates to agrimonol B, in particular to application of agrimonol B as an inducer for inducing TFEB nuclear translocation and application of agrimonol B in treating diseases of cholesterol accumulation and phospholipid accumulation related to TFEB nuclear translocation.

Background

Modulation of cellular cholesterol levels is essential for normal cellular function and development. Cholesterol levels within a cell are regulated, in part, by the transport of cholesterol between various compartments and membranes. The proper distribution of cholesterol between various cell membranes is important for many biological functions, such as signal transduction and membrane exchange. Cholesterol levels can also be modulated by trafficking extracellular receptors from the cell to the removal of cholesterol. These cholesterol transport mechanisms have been extensively studied, and defects in the regulation of cellular cholesterol levels have been linked to various diseases.

Niemann pick disease is a hereditary disease associated with lysosomal storage disorders. Currently, the effective treatment scheme for niemann pick disease is very limited, and no special treatment is available. There are four types of niemann-pick disease: A. b, C and type D. Niemann pick disease type C (NPC) is an autosomal recessive hereditary disease that causes the abnormal accumulation of cholesterol and other lipids in many types of cells.

There are two genes associated with NPC deficiency in humans, NPC1 and NPC 2. NCP1 is a multi-spanning membrane protein that normally associates with late endosomes or lysosomes, degrading organelles and hydrolyzing cholesterol esters carried into cells via lipoproteins. NPC1 deficiency may lead to decreased Autophagy and Cholesterol Accumulation, which in turn leads to severe neurodegeneration and liver dysfunction, i.e., the development of Niemann Pick disease (Dorotha Maetzel, Sovan Sarkar, Haoyi Wang, et al.genetic and Chemical Correction of Cholesterol Accumulation and Impatived Autophargym in Heapatic and Neural Cells Derived from Niemann-Pick Type C Panel-Specific iPS Cell, Stem Cell Reports,2014,2, 866-880). Due to the loss of function of NPC1 protein, cholesterol metabolism is abnormal. Recent studies have shown that abnormal cholesterol metabolism due to loss of NPC1 function is associated with abnormal autophagy regulation.

Phosphatidosis is a condition in which there is an excessive accumulation of phospholipids in body tissues. Excessive accumulation of phospholipids is thought to be associated with altered synthesis and/or metabolism of phospholipids. Phosphatidosis occurs when a patient is given a drug. For example, phosphatidic diseases can be caused when humans are amiodarone, perhexiline, fluoxetine, gentamicin.

Therefore, there is a need to develop a new effective therapeutic means for diseases caused by defective regulation of cellular cholesterol levels, such as Niemann pick disease, and for phospholipidosis.

Disclosure of Invention

In view of the above problems, the present invention provides agrimophol B, a TFEB nuclear translocation inducer, which induces TFEB translocation from the cytoplasm into the nucleus and thereby reduces intracellular cholesterol and phospholipid accumulation.

The invention provides an application of agrimonol B as a TFEB nuclear translocation inducer, wherein the chemical structural formula of the agrimonol B is shown in the specification

Furthermore, the application of the agrimophol B as a TFEB nuclear translocation inducer is clinical treatment.

Further, the clinical treatment is the treatment of diseases associated with cholesterol accumulation.

Further, the clinical treatment is for treating diseases associated with phospholipid deposition.

Further, the disease associated with phospholipid deposition is a phosphatidic disease.

Another aspect of the present invention provides the use of agrimophol B for the manufacture of a medicament for the treatment of a disease treated by induction of TFEB nuclear translocation, the agrimophol B having the chemical formula

Further, the disease is a disease associated with cholesterol accumulation.

Further, the above disease is a phospholipid accumulation-related disease.

In another aspect, the invention provides the use of agrimophol B as an inducer of autophagy, wherein agrimophol B has the chemical structural formula as shown in the specification

Defining: nuclear translocation, which is the translocation of the nuclear molecule of the present invention, is the entry of cytoplasmic material into the nucleus.

Herba et Gemma Agrimoniae and agrimophol B, wherein the medicinal source of herba et Gemma Agrimoniae is Agrimonia pilosa Ledeb of Rosaceae, and herba et Gemma Agrimoniae is mainly produced in Zhejiang, Jiangsu, Hubei, etc. In the traditional clinical application, the agrimony has the effects of astringing to stop bleeding, stopping dysentery, preventing malaria and tonifying deficiency, and is traditionally used for treating bleeding, diarrhea, dysentery, malaria, chills and fever and weakness and internal injury. The agrimophol B is a polyphenol compound from the traditional Chinese medicine agrimony and has a phloroglucinol structure.

Cholesterol is also known as cholesterol. A derivative of cyclopentane multi-hydrogen phenanthrene. Cholesterol is widely present in animals, most abundantly in brain and nerve tissues, and is also present in kidney, spleen, skin, liver and bile. The solubility of the product is similar to that of fat, and the product is insoluble in water and easily soluble in solvents such as ether and chloroform. Cholesterol is an essential substance indispensable to animal tissue cells, not only participates in the formation of cell membranes, but also is a raw material for the synthesis of bile acids, vitamin D and steroid hormones.

TFEB (transcription factor EB), a member of the Mi T/TFE family of the leucine zipper b HLH-Zip transcription factor family. TFEB has been reported to be involved in regulation of lysosomal synthesis and function, regulation of autophagy, and association with various diseases such as neurodegenerative diseases, cancer, and lysosomal storage disorders. Recently, it has been reported that overexpression of TFEB can prevent MPTP-induced apoptosis of dopamine neurons; activation of TFEB can alleviate abnormal accumulation of alpha-synuclein in brain tissue and damage to nerve cells caused by oxidative stress and inflammation.

LC3(Light Chain 3) is a marker protein on autophagosome membranes. Two forms of LC3 protein, LC 3-I and LC 3-II, exist in cells. The C-terminus of newly synthesized LC3 in the cell was cleaved by Atg4 protease to become the cytosolic soluble form of LC 3-I. After autophagosome formation, LC 3-I is cut and modified by ubiquitination processing, coupled with Phosphatidylethanolamine (PE) on the surface of autophagosome membrane, becomes LC 3-II in membrane-bound form and is positioned on the inner membrane and the outer membrane of autophagosome. Unlike some other Atg proteins localized on the membrane of autophagosomes (which only play a role at a certain stage of the autophagy process), LC 3-ii remains stably on the membrane of autophagosomes until fusion with lysosomes and is thus used as a marker molecule for autophagosomes. The content of LC 3-II or the ratio of LC 3-II/LC 3-I is in direct correlation with the number of autophagosomes and reflects the autophagy activity of the cells to some extent.

The invention has the beneficial effects that:

1. the invention discovers for the first time that agrimonol B can induce TFEB nuclear translocation, namely, TFEB is induced to be transferred from cytoplasm to nucleus, so that the agrimonol B is combined with genes to play a role of transcription factors to cause a series of downstream changes.

2. The invention proves that agrimonol B can induce autophagy of cells. Autophagy is a process in which lysosomes degrade the cell contents, and is the most effective example of recycling of the cell contents, and autophagy has the function of regulating the cell energy and helps to maintain the positive balance of the cell energy. The role of autophagy in cell metabolism has been its primary function. In addition to decomposing proteins and supplementing amino acids in cells, the autophagy of the cells is helpful for mobilization and hydrolysis of lipid and glycogen in the cells, and the number and the function of a mitochondrial network are controlled through the form of autophagy of the mitochondria, so that the substance metabolism, the energy metabolism and the cell renewal of the cells are finally realized.

3. The results of the invention prove that the agrimonol B can reduce the accumulation of cholesterol and phospholipid in cells, and thus can be used as a potential drug for treating related diseases caused by the accumulation of cholesterol and phospholipid.

Drawings

FIG. 1 shows the chemical structure of agrimophol B;

FIG. 2A is a photograph showing fluorescent images of TFEB protein in U2OS-TFEB cells after treatment of agrimophol B at different concentrations for 4 hours;

FIG. 2B shows the efficiency of agrimonol B in inducing TFEB nuclear translocation;

FIG. 2C shows the TFEB fluorescence intensity of agrimonol B induced TFEB nuclear translocation nucleus region;

FIG. 3A is a photograph of fluorescent images of GFP-LC3 and mCherry-LC 3;

FIG. 3B shows the concentration effect of agrimophol B treatment on GFP-LC 3;

FIG. 3C shows the concentration effect of Agrimonol B treatment on mCherry-LC 3;

FIG. 3D shows an assay for the effect of agrimophol B on GFP-mCherry-LC3 protein formation;

FIG. 3E shows the effect of agrimophol B on the number of U2OS-mCherry-Green-LC3 cells at 4 hours;

FIG. 4A is a photograph of fluorescence imaging of cholesterol Filipin III marker in GM03123 cells after 48 hours of agrimonol B treatment;

FIG. 4B is a photograph of CMRA marker GM03123 cytoplasmic fluorescence imaging after 48 hours of agrimophol B treatment;

FIG. 4C shows the cholesterol spot analysis of agrimophol B effect for 48 hours;

FIG. 4D shows the Filipin III fluorescence intensity analysis of agrimophol B at 48 hours;

FIG. 4E shows the effect of agrimophol B treatment for 48 hours on the number of GM03123 cells;

FIG. 5A is fluorescence imaging of GM03123 cell cholesterol Filipin III marker after 72 hours of agrimophol B treatment;

FIG. 5B is CMRA marker GM03123 cytoplasmic fluorescence imaging after 72 hours of agrimophol B treatment;

FIG. 5C shows the cholesterol spot analysis of agrimophol B effect for 72 hours;

FIG. 5D shows the fluorescence intensity analysis of Filipin III by agrimol B after 72 hours of action;

FIG. 5E shows the effect of agrimophol B treatment for 72 hours on the number of GM03123 cells;

FIG. 6A is a photograph of the phospholipid and nuclear fluorescence images of HepG2 cells after agrimophol B treatment;

FIG. 6B shows the effect of agrimophol B concentration on the concentration of HepG2 cell phospholipids;

FIG. 6C shows the effect of phospholipid counts on agrimophol B concentration;

FIG. 6D shows the effect of HepG2 cell number on the concentration of agrimophol B.

Detailed Description

The technical solution of the present invention will be described in detail with reference to the following examples. Reagents, instruments and equipment used in the examples are publicly available.

The experimental apparatus used in the experiment mainly comprises: imageexpress high-content fluorescence imager; cellomics ArrayScan high content fluorescence imaging system.

Experimental samples: agrimonol B

Example 1: formulating compounds

(1) Preparing agrimophol B: weighing agrimonol B powder, dissolving in DMSO to prepare 10mmol/L stock solution, and storing at-20 deg.C. In vitro cell experiments were performed after dilution in DMEM complete medium.

(2) Rapamycin (rapamycin) was formulated: rapamycin powder was weighed and dissolved in DMSO to prepare 10mmol/L stock solution, which was stored at-20 ℃. In vitro cell experiments were performed using DMEM complete medium diluted to 1. mu.M.

(3) Preparing a Torin 1: torin1 powder was weighed out to prepare a stock solution of 1mmol/L, and stored at-20 ℃. In vitro experiments were performed with DMEM complete medium diluted to 250 nM.

(4) Preparing a SAHA solution: SAHA powder was weighed to prepare 100mmol/L stock solution and stored at-20 ℃. In vitro experiments were performed after dilution with DMEM complete medium.

Example 2: cell culture

U2OS-TFEB-EGFP cells were cultured in DMEM complete medium, and when the growth was good, the cells were seeded at 11,000 cells/well on a 96-well black-wall transparent bottom plate and cultured overnight. The next day, the test compound was added and after 4h incubation the TFEB nuclear translocation was examined.

The U2OS-mCherry-EGFP-LC3 stable expression cell line is cultured in a puromycin-containing culture medium of 2 mug/mL until the growth state is good, 6500 cells/well are inoculated on a 96-well black-wall transparent bottom plate, and the cell line is cultured overnight in a DMEM complete culture medium without puromycin. The next day, four hours after the test compound was added, the expression level of LC3 was measured.

GM03123 cells were cultured in EMEM medium containing 10% fetal bovine serum. To logarithmic growth phase, the cells were seeded at 2400 cells/well in a 96-well black-wall clear bottom plate and cultured overnight. The next day, the test compound was added and incubated for 48 hours and 72 hours for fluorescent labeling, respectively.

HepG2 was cultured in DMEM complete medium containing 10% fetal bovine serum, 100U/mL streptomycin sulfate and 100. mu.g/mL penicillin. The next day of recovery, fresh medium was replaced. Cell passage is carried out every 2-3 days, and the drug activity research is carried out when the cell growth state is good. The number of cell passages is within 14 passages.

Example 3: research on TFEB nuclear translocation induced by agrimonol B

Agrimonol B was diluted to concentrations of 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, and 1.5625. mu.M, respectively, and 20. mu.L of each was pipetted and added to U2OS-TFEB-EGFP cells containing 180. mu.L of the medium, respectively, so that the final concentrations were 20. mu.M to 156.25nM, respectively. In parallel experiments, 1. mu.M rapamycin and 250nM Torin1 were used as positive controls. Cells from the group without drug treatment were used as negative controls. Meanwhile, culture media without glucose, glucose and glutamine were used as control for nutrient starvation. After 4h of drug incubation, cells were fixed with 4% paraformaldehyde. Cell nuclei were labeled with 10. mu.g/mL Hoechst 33342, and 100. mu.L of fluorescent dye was incubated with the cells for 15 minutes. The cells were rinsed 3 times with PBS and TFEB and nuclear fluorescence pictures were taken using MD ImageXpress. As shown in fig. 2, agrimophol B induced nuclear translocation of TFEB. The inventor finds that the agrimonol B can remarkably activate TFEB nuclear penetration at 10 mu M, and the cell positive rate is close to 100%. Further studies found that as the concentration decreased, its effect of promoting TFEB nuclear translocation decreased rapidly. The concentration setting is optimized, and the concentration is decreased in sequence according to 2 mu M, so that the change of the hairyvein agrimonol B induced TFEB nuclear translocation concentration effect can be clearly observed, an inflection point appears at about 5 mu M, and the hairyvein agrimol B does not significantly induce the cell number loss in the evaluated concentration range. The application shows that the agrimophol B activates the TFEB nuclear translocation action for the first time, and has the potential effect of regulating cell metabolism. Phosphatidopathy is characterized by the accumulation of phospholipids into lysosomes. Research shows that the accumulation of the phospholipid activates lysosome-cell nucleus signal conduction mediated by TFEB, can promote lysosome biosynthesis, autophagy, endocytosis and the like, and is possibly used as a protective reaction for degrading the accumulated phospholipid. Among them, TFEB is a key regulator of the autophagy-lysosome system and can reduce drug-induced phospholipid stacking when TFEB is overexpressed. This finding indicates that TFEB has a protective effect on phospholipid accumulation.

Example 4: agrimonol B induces LC 3-mediated autophagy flux

Agrimonol B was prepared in DMEM complete medium into working solutions at 100. mu.M, 30. mu.M, 10. mu.M, 3. mu.M, 1. mu.M, and 0.3. mu.M, respectively, and 20. mu.L of the working solution was pipetted and added to U2OS-mCherry-EGFP-LC3 cells containing 180. mu.L of the medium. After 4 hours of incubation, nuclei were labeled with 10. mu.g/mL Hoechst 33342 solution. After 15 minutes of incubation, after 3 washes with PBS, mCherry-LC3, GFP-LC3 and nuclear information were collected using the ImageXpress Micro capacitive High-Content Imaging System platform. The average number of mCherry-LC3 and GFP-LC3 in each cell, the number of mCherry-EGFP-LC3 and the change of the cell number are analyzed by a Custom module method. FIG. 3A shows the image of LC3 fluorescence when agrimophol B is acted for 4h, while Vehicle (blank) shows the negative control group, and the result shows that the negative control group has a certain number of LC3 cells distributed. Torin1(250nM) represents the positive control, and Torin1(250nM) significantly enhanced GFP-LC3 and mCherry-LC3 counts after treatment. Agrimonol B significantly enhanced the formation of GFP-LC3 and mCherry-LC3, as well as co-localized LC 3. FIG. 3B is a graph showing the analysis of GFP-LC3 fluorescence intensity data, which shows that agrimophol B dose-dependently promotes the formation of GFP-LC3, and under the action of 10. mu.M, the amount of GFP-LC3 formed is about 8 times that of the negative control. At high concentrations (6.67-10. mu.M), GFP-LC3 accumulation increased slightly with longer duration of action. At low concentrations (1.32-4.44. mu.M), GFP-LC3 formation decreased with increasing duration of action. FIG. 3C shows the mCherry-LC3 fluorescence intensity data analysis chart, after the action of agrimonol B, the expression of mCherry-LC3 is obviously increased, and the accumulation amount is obviously more when the treatment is carried out for 4 hours than when the treatment is carried out for 2 hours. When the drug incubation time is increased to 6h, the expression of mCherry-LC3 does not continuously increase, but slightly decreases. It is shown that agrimophol B significantly promotes early autophagosome formation during autophagy. FIG. 3D is a chart of mCherry-EGFP-LC3 Area Ratio analysis data, showing the evaluation of the direction of autophagy flow using the mCherry-LC3 to GFP-LC3 Area Ratio (mCherry/GFP Area Ratio). The ratio of the total LC3 amount of the cells in the normal control group to the cytoplasmic LC3 amount is between 1.6 and 2.0, which indicates that the cells have a certain level of autophagy flow. The ratio of the Torin1 treatment group at different times is equivalent to that of the negative control group, and the autophagy effect of the cells is maintained. After the high concentration treatment of the agrimophol B for 2-6h, the level of mCherry/GFP is slightly reduced, but still higher than 1, which indicates that a certain amount of autophagolytic enzyme still forms. When the concentration is reduced to 4.4. mu.M, the ratio is restored to about 1.6. There was no significant change in the cumulative amount of LC3 within 4-6h of agrimophol B treatment, indicating that there was no sustained increase in autophagosome formation beyond 4h of duration of action. FIG. 3E shows the effect of Agrimonol B on the number of U2OS-mCherry-EGFP-LC3 cells. The number of the cells of the negative control group is not obviously different from that of the positive control group, the number of the cells of the positive control group is reduced along with the increase of the action time, and the number of the cells is reduced along with the reduction of the concentration after the agrimonol B treatment, wherein the number of the cells of the drug treatment for 4h is higher than that of the drug treatment for 2h and 6 h.

Example 5: agrimonol B has effect of reducing cholesterol accumulation of GM03123 cell

Agrimonol B was diluted to a concentration of 300. mu.M, 100. mu.M, 30. mu.M, 10. mu.M, 3. mu.M, 1. mu.M, 0.3. mu.M, 0.1. mu.M, respectively, and 20. mu.L of the diluted solution was added to a medium containing 180. mu.L of the diluted solution so that the final concentration was sequentially diluted from 30. mu.M in 3-fold gradient. After 48 hours and 72 hours of compound incubation, respectively, whole cells were labeled by incubation with 5 μ M CMRA at 37 ℃ for 30 minutes. Cholesterol in the cells was labeled with 25. mu.g/mL Filipin fluorescent dye. Fluorescence information was collected using Thermo Scientific HCS VTI. Cells were identified by CMRA, cholesterol fluorescence markers were identified by Spot, and the average number of spots per cell (Spot count/cell) and Spot Intensity (Spot Intensity/cell) were analyzed. FIGS. 4A and 4B show the cholesterol fluorescence of GM03123 cells after 48h treatment with agrimophol B at various concentrations, labeled with Filipin and CMRA-labeled cell number. FIGS. 5A and 5B show the cholesterol fluorescence of GM03123 cells from NPC1 mutant patients as a model of cholesterol stacking cells after 72h of treatment with Agrimonol B at various concentrations, labeled with Filipin and CMRA-labeled cell numbers. GM05659 is derived from healthy human cells, 0.1% DMSO is added as a negative control group, 10 μ M SAHA is used as a positive control group, and agrimonol B with different concentrations acts on GM03123 cells for 48h and 72h respectively, and then Filipin is used for analyzing the content of free cholesterol in the cells and the number of the cells. The highest inhibition rate is achieved when the agrimophol B is treated for 48h at the concentration of 10 mu M, and the highest inhibition rate is 3.3 mu M when the agrimophol B is treated for 72 h. The effect of 30 μ M agrimonol B treatment for 48h and 72h was weaker than that of the low concentration group, and a rebound concentration effect appeared. The concentration response curves for both treatment time groups appeared in a U-shape with IC50 of 0.94. mu.M and 0.68. mu.M, respectively. Agrimophol B treatment for 48h showed no strong cytotoxicity and the cell number tended to decrease slightly. As the duration of action was extended to 72h, a more pronounced loss of cell number occurred in the 10-30 μ M treated group (FIGS. 3-6). The study shows that the agrimonol B has the function of remarkably improving the cholesterol deposition, and the better condition is that the treatment is carried out for 72 hours at the speed of 3 mu M.

Example 6: research on inhibition effect of agrimonol B on phospholipid accumulation

The 96-well cell culture plates were incubated with colagen I (30. mu.L/well, 100. mu.g/mL) at 37 ℃ for 30min and rinsed 3 times with PBS. HepG2 cells were seeded at a density of 5,500 cells/well in 96-well plates containing 100. mu.L of DMEM complete medium per well. The cell culture plates were placed in a 37 ℃ cell incubator and incubated overnight.

The next day, a phospholipid detection fluorescent dye was prepared according to the procedure recommended by the kit. LipidTeXTM Red phospholiposis fluorescent reagent was diluted 500-fold with DMEM complete medium to prepare 2X fluorescent dye, and the 2X fluorescent dye was filtered through a 0.2 μm filter. The inducing solution was diluted with the fluorescent dye to 20. mu.M Amiodarone (Amiodarone) or 60. mu.M propranolol (propranolol), and the resultant was applied to a cell culture plate at 50. mu.L/well as an inducer for cell phospholipid formation.

Meanwhile, a 2X compound solution was prepared and added to the cells at 50. mu.L/well so that the volume of the cell fluid became 100. mu.L/well. When the activity is verified, the concentration of the agrimonol B is respectively as follows: 10. mu.M, 8. mu.M, 6. mu.M, 4. mu.M, 2. mu.M, 0. mu.M.

The control groups were: the vehicle control group (vehicle) contained phospholipid inducer, and the blank control group contained no phospholipid inducer. All cells were incubated with fluorescent dye. Each compound was replicated in 3 replicates. The cell culture plate was placed in a 37 ℃ cell culture chamber for 48 h.

Cell fixation

(1) The cell culture medium was discarded, 100. mu.L of preheated 4% PFA was added to each well, and the cells were fixed by leaving it at room temperature for 20 min.

(2) The cells were rinsed 3 times with PBS, 100. mu.L of PBS was added each time.

(3) mu.L of Hoechst 33342 solution (10. mu.g/mL) was added to each well and incubated at room temperature for 15min to label the nuclei.

(4) The cells were rinsed 3 times with PBS, 100. mu.L of PBS was added each time.

(5) 100 μ L of PBS was added to each well for fluorescence imaging.

Phospholipid fluorescence analysis

Fluorescent signals are collected on a colloid ArrayScan TVI high content platform, a 10X objective lens is adopted, and a cell nucleus signal Ex/Em: 386/461 nm; the phospholipid signal was collected on an Alexa Fluor 594/Texas Red filter. 6 fields per well were collected.

The number of cells per well (Cell count), the average number of phospholipids per Cell (Spot count/Cell) and the fluorescence Intensity of phospholipids (Spot Intensity/Cell) were analyzed by Spot Detection analysis. Fig. 6 shows the inhibitory effect of agrimophol B on propranolol-induced phospholipid formation, and fig. 6A shows intracellular phospholipid and nuclear fluorescence pictures of HepG2, and the results show that agrimophol B significantly reduces intracellular phospholipid content and significantly reduces cell number. The cell inhibiting rate is about 75% when the cell inhibiting effect is stable on HepG2 cells at 4-10 mu M, and the cell number is close to that of a negative control group at 2 mu M. FIGS. 6B, 6C and 6D show the concentration effect curves of HepG2 cell number, phospholipid droplet number and fluorescence intensity after 48h of agrimophol B treatment, showing good concentration-dependent effect relationship within 10. mu.M, complete inhibition of phospholipid production within 4-10. mu.M, partial phospholipid droplets at 2. mu.M, but relatively little fluorescence. Although the number of cells is reduced, there is still some space between the content of phospholipids and the number of cells. The results of this study further confirmed that agrimophol B can inhibit drug-induced phospholipid production.

The technical solutions described above only represent the preferred technical solutions of the present invention, and some possible modifications to some parts of the technical solutions by those skilled in the art all represent the principles of the present invention, and fall within the protection scope of the present invention.

Claims

1. An application of agrimonol B as a TFEB nuclear translocation inducer in preparing a medicament for treating C-type Niemann pick disease and/or treating phosphatide disease;

wherein the chemical structural formula of the agrimophol B is shown as the following formula

2. An application of agrimonol B as an inducer of autophagy in preparing a medicament for treating C-type Niemann pick disease and/or treating phosphatide disease;