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Application of ingenol and its derivatives in enhancing lysosome production

A lysosome and drug technology, applied in the field of application of ingenol HEP14 and its derivatives in enhancing lysosome production, can solve the problem that the signaling pathway has not been reported yet

Active Publication Date: 2019-10-18
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, except for the signaling pathway that senses nutritional status that can regulate lysosome production has been confirmed, other signaling pathways have not been reported

Method used

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  • Application of ingenol and its derivatives in enhancing lysosome production
  • Application of ingenol and its derivatives in enhancing lysosome production
  • Application of ingenol and its derivatives in enhancing lysosome production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1, Preparation and identification of HEP14 and its derivatives

[0085] 1. Preparation and identification of HEP14 and HEP15

[0086] (1) Preparation of HEP14 and HEP15

[0087] With dried Euphorbia peplus Linn. whole herb (9.5 kg), heat reflux extraction with 95% ethanol for 4 times (extraction time is successively 5, 4, 3, 3 hours) and then filter, combine the filtrates, and reclaim the ethanol Then add water to suspend, and then extract with petroleum ether and chloroform sequentially, each time extracting 4 times with equal volume, and recover the extraction solvent under reduced pressure to obtain petroleum ether extraction fraction (about 299 g) and chloroform extraction fraction (about 17 g). Then the petroleum ether extraction part was subjected to 200-300 mesh silica gel column chromatography, gradient elution with petroleum ether-acetone mixed solvent (petroleum ether / acetone: 1:0→0:1), and detected by thin layer chromatography (TLC). Combined, 6 fra...

Embodiment 2

[0107] Example 2, HEP14 induces lysosome generation

[0108] 1. Lyso Tracker Red staining method to detect the regulation of HEP14 on lysosome production

[0109] Treat HeLa cells with HEP14 compound, and then use LysoTracker Red staining method to observe the coloring of LysoTracker to observe the changes in the number of lysosomes. The specific operation is as follows:

[0110]HeLa cells were treated with different concentrations of HEP14 compounds (0 μM, 10 μM, 20 μM, 40 μM, and the solvent was DMSO). After 3 hours, the cells treated with different treatments were placed in 0.3 μM Lyso Tracker Red DND-99 (product of Invitrogen, Inc., The product catalog number is L7528) culture medium for 30min, and then on the one hand, direct laser confocal observation and imaging; on the other hand, adopt PBS suspension cells to sort through flow cytometer FACSAria II, and finally use FlowJo software for statistics, Get the number of lysosomes. In the experiment, Torin1, an mTORC1 inh...

Embodiment 3

[0119] Example 3, HEP14 compound induces TFEB protein to enter the nucleus

[0120] 1. Detection of the effect of HEP14 on the expression of TFEB and TFE3 target genes

[0121] Hela cells were treated with HEP14 at a concentration of 20 μM for 3 hours, and the effect of HEP14 on the expression of TFEB and TFE3 target genes in cells was detected with Hela cells not treated with HEP14 (replaced with an equal amount of DMSO) as a control. The specific operation is as follows: the RNA in the cell sample is extracted with Trizol (Invitrogen) and chloroform and then quantified, 2 μg of each sample is taken as a template, and cDNA is synthesized with ImProm-II Reverse Transcription system (Promega), and the subsequent PCR reaction is carried out in MX3000P (Agilent Technologies) instrument, each sample was repeated three times, and the internal reference gene was GAPDH. Data analysis using 7900HT Fast Real-Time PCR system software.

[0122] The results showed that after HEP14 treat...

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Abstract

The invention discloses ingenol and application of a derivative of ingenol in enhancement of generation of lysosome. The application specifically comprises optional application of HEP14 or the derivative thereof in the following applications: induction of generation of the lysosome; preparation of drugs for induction of generation of the lysosome; and preparation of drugs for treating and / or preventing lysosome functional disturbance related diseases. HEP14 can activate PKC alpha and PKC delta, on one hand, transcription factors TFEB are activated through two parallel signal pathways, and on the other hand, transcription inhibitors are inactivated and are finally used as 'molecular switches' to control generation of the lysosome. Activation of PKC causes inactivation of GSK3 beta, then transfer of TFEB dephosphorylation to cell nucleus is caused, meanwhile, PKC which is in an activated state can further activate MAPK kinase JNK2 and p38 and phosphorylate ZKSCAN3, and ZKSCAN3 can be transferred to cytoplasm from cell nucleus. HEP14 does not affect activity of mTORC1, therefore, cell metabolism balance is not disturbed, and the ingenol is a perfect drug for treating lysosome related diseases possibly.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the application of ingenol and its derivatives in enhancing the production of lysosomes, in particular to an ingenol HEP14 (5β-O-angeloyl-20-deoxyingenol , 5-O-angeloyl-20-deoxyingenol) and its derivatives in enhancing lysosome production. Background technique [0002] Lysosomes are a class of acidic single-membrane organelles responsible for the removal of metabolic substrates. Dysfunction of lysosomal function causes lysosomal storage diseases (LSDs) and neurodegenerative diseases. Increased lysosome production can promote cell degradation of damaged organelles and denatured protein aggregates, and promote lipid metabolism, thereby avoiding related diseases. At the transcriptional level, the production of lysosomes is mainly regulated by two transcription factors, TFEB and TFE3, which up-regulate the expression of lysosomes and autophagy-related genes through the CLEAR (Coordinated...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/22A61K31/015A61K31/23A61P25/28A61P25/00A61P25/14A61P25/16
Inventor 杨崇林郝小江李洋徐猛丁骁晏晨邸迎彤穆淑珍蹇友理宋智琴郭伟翔
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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