CN109620839A - Application of the Specnuezhenide as cell autophagy inducer - Google Patents
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Abstract
The embodiment of the invention discloses Specnuezhenides to prepare the application in cell autophagy inducer or pharmaceutical composition.The discovery of the autophagy inducing effect of the Specnuezhenide of the embodiment of the present invention has it to be developed at a kind of new novel autophagy derivant, is applied in the treatment or adjuvant treatment of disease, such as the adjuvant treatment of bacterial infective diseases, in the adjuvant treatment of tumour.
Description
Technical field
The present embodiments relate to pharmaceutical technology fields, and in particular to a kind of cell autophagy inducer and application.
Background technique
Autophagy refers to the process of that intracellular long-lived proteins and impaired organelle are degraded through lysosomal pathway,
It is phenomenon specific to eukaryocyte.High temperature, anoxic, starvation etc. stress when generate autophagy can help cell resist these it is unfavorable because
Element plays cytoprotection.But autophagy excessive activation or not will lead to cell death then in due course, i.e. autophagy cell is dead
Die (autophagic cell death).Existing research shows imbalance and tumour, neurodegenerative disease, the angiocarpy of autophagy
The diseases such as disease and infection are closely related.
Occur stress when, cell autophagy can prevent the accumulation of toxic or carcinogenic injury protein matter and organelle, suppression
Cell carcinogenesis processed；However tumour is once being formed, cell autophagy provides richer nutrition for cancer cell, promotes tumour growth.To the greatest extent
Pipe autophagy low expression in most tumours prompts autophagy that may play the role of inhibiting tumour growth.But on research shows that
Autophagy is adjusted again such that the invasion and chemotherapeutics repellence of tumour cell enhance.Autophagy is in the case where chemotherapy or radiotherapy
Tumour cell can be helped to tide over the difficulty of Metabolic stress in the case where apoptosis defect.Metabolic stress is not only present in chemicotherapy
Tumour cell in, it is insufficient or DISTANT METASTASES IN is into the organ for lacking nutrition can similarly see in tumour cell blood supply.Cause
This, autophagy induction is formed in tumour to be of great significance in early days.
In antibacterial research, autophagy is the important link of innate immunity and specific immune response.Autophagy is anti-thin
Verticillium toxin, the antigen deduction ability for enhancing MHC II class molecule have played important function in terms of removing pathogen.
Currently, common novel autophagy derivant mainly have rapamycin, Bredeldin A, Carbamazepine,
Xestospongin B/C,N-Acetyl-D-sphingosine.The existing alternative type of novel autophagy derivant is less, more
For the compound molecule of synthesis, need to be developed new novel autophagy derivant.
Summary of the invention
For this purpose, the embodiment of the present invention, which provides Specnuezhenide, is preparing answering in cell autophagy inducer or pharmaceutical composition
With.
To achieve the goals above, embodiments of the present invention provide the following technical solutions:
Specnuezhenide is preparing the application in cell autophagy inducer or pharmaceutical composition.
Preferably, application of the Specnuezhenide in induction gland cancer mankind alveolar substrate epithelial cell A549 autophagy.
Preferably, the inducer is peroral dosage form or injection type.
Preferably, described pharmaceutical composition further includes the effective component of sterilization or treatment tumour.
Preferably, the effective component of the treatment tumour includes cis-platinum or taxol.
Preferably, the illness of described pharmaceutical composition treatment includes bacterium infection relevant to cell autophagy and tumour.
Preferably, the tumour is leukaemia, lung cancer, colon cancer, kidney, prostate cancer, oophoroma, the cancer of the brain, mammary gland
Cancer, cancer of pancreas, cervix cancer, gastric cancer, liver cancer, sarcoma or melanoma.
The bacterium infection include the staphylococcuses such as MRSA, Staphylococcus aureus or Diplococcus pneumopniae,
The gram-positive bacterias such as bacillus anthracis, corynebacterium diphtheriae, clostridium tetani.
It is formed under fluorescence microscope using GFP-mCherry-LC3 fusion protein come tracer autophagy.When cell-free autophagy,
GFP-LC3 fusion protein disperse is in endochylema；When autophagosome formation, GFP-LC3 fusion protein is indexed into autophagosome film, in fluorescence
Multiple bright green fluorescence spots are formed under microscope, a spot is equivalent to an autophagosome, can comment by counting
The active height of valence autophagy.
Specnuezhenide in the embodiment of the present invention is leaf and the fruit of glossy privet (female of Oleaceae glossy privet Ligustrum lucidum
Loyal fruit) extract, molecular formula C31H42O17.Its structural formula is as shown in Figure 1.
In the embodiment of the present invention, autophagy refers to intracellular long-lived proteins and impaired organelle through lysosome way
The process that diameter is degraded is phenomenon specific to eukaryocyte.High temperature, anoxic, starvation etc. stress when generate autophagy and can help carefully
Born of the same parents resist these unfavorable factors, play cytoprotection.But autophagy excessive activation or not in due course, then it is dead to will lead to cell
It dies, i.e. autophagy cell death (autophagic cell death).Existing research shows the imbalance and tumour, nerve of autophagy
The diseases such as degenerative disease, cardiovascular disease and infection are closely related.
It has the advantages that according to embodiments of the present invention
The discovery of the autophagy inducing effect of the Specnuezhenide of the embodiment of the present invention, Specnuezhenide are native compound molecule,
Have it to be developed at a kind of new novel autophagy derivant, is applied in the treatment or adjuvant treatment of disease, such as bacterium infection disease
The adjuvant treatment of disease, in the adjuvant treatment of tumour.
Detailed description of the invention
It, below will be to embodiment party in order to illustrate more clearly of embodiments of the present invention or technical solution in the prior art
Formula or attached drawing needed to be used in the description of the prior art are briefly described.It should be evident that the accompanying drawings in the following description is only
It is merely exemplary, it for those of ordinary skill in the art, without creative efforts, can also basis
The attached drawing of offer, which is extended, obtains other implementation attached drawings.
Fig. 1 is the Specnuezhenide schematic arrangement of the embodiment of the present invention；
Fig. 2 is the confocal microscopy detection of the Specnuezhenide 0.34ug/mL processing group of the embodiment of the present invention
The schematic diagram of fluorescence, wherein Fig. 2A is DAPI colored graph, and Fig. 2 B GFP fluorogram, Fig. 2 C is mCherry fluorogram, and Fig. 2 D is
GFP and the fused fluorogram of mCherry；
Fig. 3 is that the confocal microscopy detection of the Specnuezhenide 1.1ug/mL processing group of the embodiment of the present invention is glimmering
The schematic diagram of light, wherein Fig. 3 A is DAPI colored graph, and Fig. 3 B is GFP fluorogram, and Fig. 3 C is mCherry fluorogram, and Fig. 3 D is
GFP and the fused fluorogram of mCherry；
Fig. 4 is that the confocal microscopy detection of the Specnuezhenide 3.4ug/mL processing group of the embodiment of the present invention is glimmering
The schematic diagram of light, wherein Fig. 4 A is DAPI colored graph, and Fig. 4 B is GFP fluorogram, and Fig. 4 C is mCherry fluorogram, and Fig. 4 D is
GFP and the fused fluorogram of mCherry；
Fig. 5 is that the confocal microscopy of the MRSA processing group of the embodiment of the present invention detects the schematic diagram of fluorescence,
In, Fig. 5 A is DAPI colored graph, and Fig. 5 B is GFP fluorogram, and Fig. 5 C is mCherry fluorogram, and Fig. 5 D is that GFP and mCherry melts
Fluorogram after conjunction；
Fig. 6 is that the confocal microscopy of the positive controls rapamycin of the embodiment of the present invention detects fluorescence
Schematic diagram, wherein Fig. 6 A be DAPI colored graph, Fig. 6 B be GFP fluorogram, Fig. 6 C be mCherry fluorogram, Fig. 6 D be GFP with
The fused fluorogram of mCherry；
Fig. 7 is that the confocal microscopy of the negative control group chloroquine of the embodiment of the present invention detects the signal of fluorescence
Figure, wherein Fig. 7 A be DAPI colored graph, Fig. 7 B be GFP fluorogram, Fig. 7 C be mCherry fluorogram, Fig. 7 D be GFP with
The fused fluorogram of mCherry；
Fig. 8 is that the confocal microscopy of the embodiment of the present invention normally organized detects the schematic diagram of fluorescence, wherein
Fig. 8 A is DAPI colored graph, and Fig. 8 B is GFP fluorogram, and Fig. 8 C is mCherry fluorogram, and Fig. 8 D is after GFP is merged with mCherry
Fluorogram.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation
Content disclosed by book is understood other advantages and efficacy of the present invention easily, it is clear that described embodiment is the present invention one
Section Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not doing
Every other embodiment obtained under the premise of creative work out, shall fall within the protection scope of the present invention.
In the embodiment of the present invention, using small molecule compound Specnuezhenide as the novel autophagy derivant of small molecule.
In the embodiment of the present invention, autophagy process is observed and is detected, and cell, need to be to autophagy process after induction or inhibition
It is observed and is detected.
In the embodiment of the present invention, the monomeric compound Specnuezhenide with autophagy inducing action, with apparent autophagy
Inducing action, and it is from a wealth of sources, expand the type of induction autophagy instrument medicine, has needed to be developed new novel autophagy derivant treatment
Disease.
In the embodiment of the present invention, MOI:multiplicity of infection, as infection multiplicity.
In the bright embodiment of this law, cell autophagy is a kind of defence and protection mechanism of body, as the bacterium infection gland cancer mankind
When alveolar substrate epithelial cell A549, gland cancer mankind's alveolar substrate epithelial cell A549 can by cell autophagy bacterium for degrading, but
Some bacteriums can escape the survival that autophagy is conducive to itself to its phagocytosis, such as Methicillin-resistant Staphylococcus aureus MRSA.
In the embodiment of the present invention, autophagy stream testing principle are as follows:
Ad-mCherry-GFP-LC3B, i.e. adenovirus expressing mCherry-GFP-LC3B fusion
Protein is a kind of adenovirus that can express mCherry-GFP-LC3B fusion protein, can be used for infection cell or tissue
The detection of cell autophagy (autophagy) is carried out afterwards.
LC3 is the homologous protein of yeast autophagy key protein ATG8 in mammals.LC3 is initially analyzed and is accredited as
microtubule-associated protein 1 light chain 3(MAP1LC3).LC3 protein family include LC3A,
B, the subfamilies such as C and GABARAP, wherein the most extensive for the research of LC3B.LC3B is considered as cell autophagy signal path
The most key significant albumen.LC3B is the albumen with 125 amino acid residues, after albumen synthesis, by Atg4
Digestion and 22 amino acid proteins for losing C-terminal, to expose the glycine of C-terminal, it is named as cytoplasmatic forms by people
LC3B I.During cell autophagy, process of the glycine of C-terminal exposure by a similar ubiquitination, with ATG7
For E1 sample kinase (E1-like activating enzyme), with ATG3 for E2 sample ligase (E2-like
Conjugation enzyme), with Atg12-Atg5-Atg16 compound for E3 sample ligase (E3-like ligase), in C
It is covalently attached upper phosphatidyl-ethanolamine (phosphatidylethanolamine, PE) on the glycine of end and is as LC3B-PE
LC3B II.Different from the LC3B I of cytoplasm positioning, LC3B II is positioned at the inner membrance and outer membrane of autophagosome (autophagosome)
On.After autophagosome and lysosome fusion, the LC3B II on autophagosome outer membrane is by the digestion of Atg4 institute, and on autophagosome inner membrance
LC3B II is then degraded by the intracorporal protease of lyase.
After Ad-mCherry-GFP-LC3B infection can in target cell effective expression red fluorescent protein mCherry, green
The fusion protein of color fluorescin (GFP) and LC3B are presented bright red and green fluorescence, can be used for the inspection of cell autophagy
It surveys.
For autophagosome during with lysosome fusion, the intracorporal acidic environment of lyase will lead to GFP fluorescent quenching, this is
The cellular localization of tracking GFP-LC3B increases difficulty.MCherry is one kind from mushroom coral (mushroom coral)
Monomer red fluorescent protein be quenched in GFP by lysosomal acid environment when it carries out common label of LC3B with GFP
In the case where, the mCherry of red fluorescence can be issued and be retained because of its brilliant stability.Therefore, by merging table
Up to mCherry-GFP-LC3B albumen, autophagy process can be effectively tracked.With Ad-mCherry-GFP-LC3B adenopathy
After malicious infection cell, in the case where non-autophagy, mCherry-GFP-LC3B is under fluorescence microscope with the yellow fluorescence of disperse
(resultant effect of mCherry and GFP) form is present in cytoplasm；And in the case where autophagy, under fluorescence microscope
MCherry-GFP-LC3B is then gathered on autophagosome film, and (LC3B dot or is showed in the form of yellow spotting
punctae)；After autophagosome and lysosome fusion, shown in the form of punctation because the part of GFP fluorescence is quenched
Come.
Detection of 1 Specnuezhenide of embodiment in induction human lung cancer epithelial cell (A549) autophagy
Experimental material: Specnuezhenide, the staphylococcus aureus (MRSA) of methicillin-resistant, human lung cancer epithelial cell, i.e.,
A549 cell (is incubated at the DMEM culture medium containing 10% fetal calf serum), and DMEM is purchased from U.S. Hyclone company, product article No.
SH30022.01；Fetal calf serum is purchased from Shanghai Ji Taiyikesai Biotechnology Co., Ltd, product article No. FND500；Ad-
MCherry-GFP-LC3B adenovirus is purchased from green skies Bioisystech Co., Ltd, product article No. C3011-1ml), etc..
The foundation of autophagy stream detection model: firstly, with 5 × 10 in 96 orifice plates4/ hole is inoculated with A549 cell, every hole
100uL, culture is for 24 hours.Ad-mCherry-GFP-LC3B adenovirus infection A549 cell, by virus stock solution used with serum free medium
60 times of dilution, infects A549 cell, and 2h is infected in the hole 25uL/.Setting one group of A549 cell is not added Ad-mCherry-GFP- simultaneously
LC3B adenovirus, as blank control.In 96 orifice plates, the DMEM culture medium containing 3% serum, the hole 75uL/ are added in every hole.Continue
Culture, adenovirus infection are changed to the fresh DMEM culture medium for containing 3% serum for 24 hours, by culture medium, continue culture for 24 hours.
The foundation of MRSA infection A549 cell model: in addition to other unexpected groups of blank control group, methicillin-resistant is taken
Staphylococcus aureus MRSA bacterium solution be used for infection experiment.A549 cell is infected with 0.1 MOI, i.e., every hole is added MRSA's
Concentration is 5 × 103A/hole.The staphylococcus aureus MRSA and A549 cell of methicillin-resistant are incubated for 1h altogether.It discards containing bacterium solution
Culture medium, washed one time with PBS buffer solution, abandon PBS, the fresh DMEM culture medium for containing 3% serum, the hole 100uL/ is added.
Experimental group: Normal group: without any processing group；
Model group: only MRSA infected group；
Autophagy positive controls: rapamycin, the hole 27nM/；
Autophagy negative control group: chloroquine, the hole 50uM/；
The concentration of Specnuezhenide of the embodiment of the present invention is 0.34ug/mL；
It according to experimental group, is added pastille culture medium (drug containing DMEM culture medium, serum content 3%), cultivates 6h.Using
Confocal microscopy detects luciferase expression.
In the case where non-autophagy, mCherry-GFP-LC3B is under fluorescence microscope with the yellow fluorescence (mCherry of disperse
With the resultant effect of GFP) form is present in cytoplasm；And in the case where autophagy, mCherry-GFP- under fluorescence microscope
LC3B is then gathered on autophagosome film, and (LC3B dot or punctae) is showed in the form of yellow spotting；Work as autophagosome
After lysosome fusion, showed in the form of punctation because the part of GFP fluorescence is quenched.
Experimental result:
As shown in figure 8, LC3 albumen is covered with this cytoplasm in the form of disperse in cell, MRSA's in Normal group
Infection is so that LC3 is appeared in around nucleus with the state of disperse.
As shown in fig. 6, drug rapamycin can promote A549 cell autophagy, and LC3 albumen is with highlighted in positive controls
Point format appear in cytoplasm (in figure arrow signified)；And red fluorescence (mCherry) quantity is more than green fluorescence
(GFP), illustrate to have occurred merging for autophagosome and lysosome, the part of GFP fluorescence is quenched and is showed in the form of punctation
Out；Illustrate that the autophagy process of positive control medicine rapamycin is unobstructed.
As shown in fig. 7, drug chloroquine inhibits cell autophagy, and LC3 albumen disperse is in cytoplasm in negative control group；And
Green fluorescence (GFP) quantity is more than red fluorescence (mCherry), illustrates that autophagosome is inhibited with merging for lysosome.
As shown in Fig. 2, the Specnuezhenide 0.34ug/mL processing group of the embodiment of the present invention, LC3 albumen occur with punctate appearance
Around nucleus, and mCherry red fluorescence quantity is more than GFP green fluorescence quantity, illustrates that autophagosome and lyase has occurred
The fusion of body, autophagy process are unimpeded.
Detection of 2 Specnuezhenide of embodiment in induction human lung cancer epithelial cell (A549) autophagy
The foundation of autophagy stream detection model: firstly, with 5 × 10 in 96 orifice plates4/ hole is inoculated with A549 cell, every hole
100uL, culture is for 24 hours.Ad-mCherry-GFP-LC3B adenovirus infection A549 cell, by virus stock solution used with serum free medium
60 times of dilution, infects A549 cell, and 2h is infected in the hole 25uL/.Setting one group of A549 cell is not added Ad-mCherry-GFP- simultaneously
LC3B adenovirus, as blank control.In 96 orifice plates, the DMEM culture medium containing 3% serum, the hole 75uL/ are added in every hole.Continue
Culture, adenovirus infection are changed to the fresh DMEM culture medium for containing 3% serum for 24 hours, by culture medium, continue culture for 24 hours.
The foundation of MRSA infection A549 cell model: in addition to other unexpected groups of blank control group, methicillin-resistant is taken
Staphylococcus aureus MRSA bacterium solution be used for infection experiment.A549 cell is infected with 0.1 MOI, i.e., every hole is added MRSA's
Concentration is 5 × 103A/hole.The staphylococcus aureus MRSA and A549 cell of methicillin-resistant are incubated for 1h altogether.It discards containing bacterium solution
Culture medium, washed one time with PBS buffer solution, abandon PBS, the fresh DMEM culture medium for containing 3% serum, the hole 100uL/ is added.
According to experimental group, pastille culture medium is added, the concentration of Specnuezhenide is 1.1ug/mL, cultivates 6h.Using
Confocal microscopy detects luciferase expression.
As shown in figure 3, experimental result: the Specnuezhenide 1.1ug/mL processing group of the embodiment of the present invention, LC3 albumen is with dotted
Form appears in around nucleus, and mCherry amount of fluorescence is more than GFP amount of fluorescence, illustrates that autophagosome and lyase has occurred
The fusion of body, autophagy process are unimpeded.
Detection of 3 Specnuezhenide of embodiment in induction human lung cancer epithelial cell (A549) autophagy
The foundation of autophagy stream detection model: firstly, with 5 × 10 in 96 orifice plates4/ hole is inoculated with A549 cell, every hole
100uL, culture is for 24 hours.Ad-mCherry-GFP-LC3B adenovirus infection A549 cell, by virus stock solution used with serum free medium
60 times of dilution, infects A549 cell, and 2h is infected in the hole 25uL/.Setting one group of A549 cell is not added Ad-mCherry-GFP- simultaneously
LC3B adenovirus, as blank control.In 96 orifice plates, the DMEM culture medium containing 3% serum, the hole 75uL/ are added in every hole.Continue
Culture, adenovirus infection are changed to the fresh DMEM culture medium for containing 3% serum for 24 hours, by culture medium, continue culture for 24 hours.
The foundation of MRSA infection A549 cell model: in addition to other unexpected groups of blank control group, methicillin-resistant is taken
Staphylococcus aureus MRSA bacterium solution be used for infection experiment.A549 cell is infected with 0.1 MOI, i.e., every hole is added MRSA's
Concentration is 5 × 103A/hole.The staphylococcus aureus MRSA and A549 cell of methicillin-resistant are incubated for 1h altogether.It discards containing bacterium solution
Culture medium, washed one time with PBS buffer solution, abandon PBS, the fresh DMEM culture medium for containing 3% serum, the hole 100uL/ is added.
According to experimental group, pastille culture medium is added, the concentration of Specnuezhenide is 3.3ug/mL, cultivates 6h, uses
Confocal microscopy detects luciferase expression.
As shown in figure 4, experimental result: the Specnuezhenide 3.3ug/mL processing group of the embodiment of the present invention, LC3 albumen is with dotted
Form appears in around nucleus, and mCherry red fluorescence quantity is more than GFP green fluorescence quantity, illustrates that autophagy has occurred
Body is merged with lysosome, and autophagy process is unimpeded.
The embodiment of the present invention 1 is into embodiment 3, and as Specnuezhenide dosage increases, the cell quantity that autophagy occurs increases；
Three dosage processing groups of Specnuezhenide (0.34ug/mL, 1.1ug/mL and 3.3ug/mL) embody one in autophagy inductive effect
Fixed dose-effect relationship, with the increase of Specnuezhenide concentration, mCherry red fluorescence quantity increases more than GFP green fluorescence quantity
More, therefore, autophagy occurs for the A549 cell that Specnuezhenide can promote MRSA to infect.
The Specnuezhenide of the embodiment of the present invention is developed as novel autophagy derivant into drug therapy disease, or adjuvant treatment disease
Disease.The Specnuezhenide novel autophagy derivant of the embodiment of the present invention can also with sterilize or the effective component cooperatively group for the treatment of tumour
At pharmaceutical composition, auxiliary for treating cancer or bacterial infection disease etc..Wherein, treat tumour effective component include cis-platinum or
Person's taxol.(MA Tai,SUN Guo-Ping,LI Jia-Bin.Methods for Autophagy Detection[J]
.Prog.Biochem.Biophys.,2012,39(3):204-209.)
Wherein, the Specnuezhenide of the embodiment of the present invention is in the death of induction tumour cell autophagy or the malicious chemotherapeutic of enhanced sensitivity biology
Tumour in object antitumor action is leukaemia, lung cancer, colon cancer, kidney, prostate cancer, oophoroma, the cancer of the brain, breast cancer, pancreas
Gland cancer, cervix cancer, gastric cancer, liver cancer, sarcoma or melanoma.
Bacterial infection disease include the staphylococcuses such as MRSA, Staphylococcus aureus or Diplococcus pneumopniae,
The diseases such as pneumonia caused by the gram positive bacteria infections such as bacillus anthracis, corynebacterium diphtheriae, clostridium tetani.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Claims (8)
1. Specnuezhenide is preparing the application in cell autophagy inducer or pharmaceutical composition.
2. application as described in claim 1, which is characterized in that
Gland cancer mankind alveolar substrate epithelial cell of the Specnuezhenide in induction methicillin-resistant staphylococcus aureus infection
Application in A549 autophagy.
3. application as described in claim 1, which is characterized in that
The inducer is peroral dosage form or injection type.
4. application as described in claim 1, which is characterized in that
Described pharmaceutical composition further includes the effective component of sterilization or treatment tumour.
5. application as claimed in claim 4, which is characterized in that
The effective component of the treatment tumour includes cis-platinum or taxol.
6. application as described in claim 1, which is characterized in that
The illness of described pharmaceutical composition treatment includes bacterium infection relevant to cell autophagy and tumour.
7. application as claimed in claim 6, which is characterized in that
The tumour is leukaemia, lung cancer, colon cancer, kidney, prostate cancer, oophoroma, the cancer of the brain, breast cancer, cancer of pancreas, son
Cervical carcinoma, gastric cancer, liver cancer, sarcoma or melanoma.
8. application as claimed in claim 6, which is characterized in that
The bacterium infection includes staphylococcus, Diplococcus pneumopniae, bacillus anthracis, corynebacterium diphtheriae, tetanus bacillus infection.
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Cited By (1)
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| CN111265545A (en) * | 2020-02-18 | 2020-06-12 | 广州中医药大学(广州中医药研究院) | Composition for treating lung tumor |
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