CN105832719B - Arctigenin is adjusting the application in PP2A activity - Google Patents
Arctigenin is adjusting the application in PP2A activity Download PDFInfo
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- CN105832719B CN105832719B CN201610278529.9A CN201610278529A CN105832719B CN 105832719 B CN105832719 B CN 105832719B CN 201610278529 A CN201610278529 A CN 201610278529A CN 105832719 B CN105832719 B CN 105832719B
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- pp2a
- atg
- arctigenin
- albumen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
Abstract
The present invention provides a kind of arctigenin and adjusts the application in the active reagent of PP2A in preparation.Arctigenin can also have the following features: wherein in the application that preparation adjusts PP2A active agent, and the additional amount of arctigenin is 1~10 μm, and the activity of PP2A is improved with the increase of the additional amount of arctigenin within this range.ATG can highly combine PP2A as one and can influence its active natural small molecule compounds, have a good application prospect.
Description
Technical field
The present invention relates to a kind of arctigenins to adjust the application in PP2A activity, belongs to tcm field.
Background technique
Arctigenin (ATG) is the principal monomer compound in Chinese medicine great burdock achene.Great burdock achene is widely used in glycosuria by clinic
The treatment of disease, but its mechanism of action and useful effect ingredient are also indefinite.This research discovery ATG has apparent reduction diabetes
The effect of murine protein urine.
PP2A is serine threonine phosphorylase, adjusts most phosphorylase in eukaryocyte.In signal transduction
Cascade reaction in other phosphorylases and kinase interactions, constitute adjust macromolecular regulation downstream signal transduction.?
P65 is the critical path for leading to inflammatory reaction in diabetic kidney disease (DKD).PP2A can be by dephosphorylation P65, to send out
Wave the effect for the treatment of DKD.In addition, having research to disclose between PP2A and proto-oncogene c-Myc at present has close contact.PP2A
With adjusting subunit B56 alpha selective in conjunction with the N-terminal of c-Myc, c-Myc expression is caused to significantly reduce.It is released using shRNA
The combination of B56 α and c-Myc can lead to c-Myc overexpression, the raising and c-Myc function of c-Myc Ser62 phosphorylation level
Enhancing phenomena such as, be easy to associate PP2A according to this and take on certain crucial role in cell proliferation and differentiation regulation.ATG
Relationship between PP2A activity does not illustrate in research before.
Summary of the invention
The purpose of the present invention is to provide a kind of ATG to adjust the application in PP2A activity, is a kind of new opplication of ATG.
Present invention employs following technical solutions:
Present invention firstly provides a kind of ATG to adjust the application in the active reagent of PP2A in preparation.
Further, it is prepared into the application for adjusting PP2A active agent, is can also have the following features: wherein in ATG,
The additional amount of ATG is 1~10 μm, and the activity of PP2A is improved with the increase of the additional amount of arctigenin within this range.
The present invention also provides a kind of active methods of PP2A in raising 293T cell, it is characterised in that: including being added ATG's
Step.
Further, the active method of PP2A in raising 293T cell of the invention, can also have the following features: it
In, the additional amount of ATG is 1 μM~10 μM.
The present invention also provides a kind of application of ATG in recycling PP2A albumen, which comprises the steps of:
Step 1,100 μM~1000 μM of ATG, which is added, stimulates cell.
Step 2 is extracted cell and is crushed；
Step 3, loading simultaneously carry out polyacrylamide gel electrophoresis；
Step 4 cuts the gel-tape of part corresponding with PP2A molecular weight, recycles albumen.
Further, the application of ATG of the invention in recycling PP2A albumen can also have the following features: wherein, return
When receiving albumen, gel-tape is pulverized with grinding rod, the buffer less than 500 μ l/ block glue is wherein being added, it is quiet at 4 degrees Celsius
It sets 10 hours, then 5000-10000rpm is centrifuged 10min, contains PP2A albumen in supernatant.
Advantageous effect of the invention
ATG of the invention is adjusting the application in PP2A activity, provides ATG and is adjusting the new application in PP2A activity,
ATG can highly combine PP2A as one and can influence its active natural small molecule compounds, have a good application prospect.
Detailed description of the invention
Fig. 1 is the result figure of DARTS-western hybridization.
Fig. 2 is the post-stimulatory PP2A activity figure of ATG of various concentration.
Specific embodiment
Below in conjunction with the specific embodiment technical solution that the present invention will be described in detail.
1, ATG stimulates the mass spectral analysis of 293T
Using 293T cell, with M-PER mammalian protein extraction buffer
(78501ThermoFisher) and inhibitors of phosphatases (50mM NaF, 10mM β-glycerophosphate, 5mM sodium
Pyrophosphate, 2mM Na3VO4) digestion 297T cell.10X TNC buffer (500mM is added in cell pyrolysis liquid
Tris-HCl pH 8.0,500mM NaCl, 100mM CaCl2) and egg is measured with bradford method (500-0006 Bio-rad)
White matter concentration.Add in cell pyrolysis liquid or ATG is not added, adds and attempt to have a strong smell 300 μM when ATG, be placed on cultivate 1h at room temperature together.With
It is placed at room temperature in the pronase at 1:1000 (10165921001, Roche) that cell pyrolysis liquid is put into various concentration afterwards
20 minutes crack protein matter.Two groups of lysate row Mass Spectrometer Method comparing differences, the result is shown in Figure 1.
Mass spectrum comparison result after Fig. 1: ATG intervention
Analysis mass spectral results are shown, are tubulins in conjunction with 2 most albumen, due to intracellular canaliculus albumen itself
Content is very high, so it is considered that being nonspecific.And due under normal condition content of the PP2A in cell it is very low, so
It is considered that the Percentage bound of ATG and PP2A is very high.
2, western blot experiment of the ATG in conjunction with PP2A.
ATG stimulates 293T cell.Select pronase optium concentration for 1:1000 after, we have selected various concentration
Can ATG stimulates 293T cell, increasing and increase with ATG concentration to observe the binding capacity of ATG and PP2A.According to ATG's
Irritaiting concentration difference is divided into 3 groups: DMSO, 100 μM, 300 μM, 1000 μM.The concentration that pronase is added is 1:1000.Therefore
Western Blot mono- is divided into 5 groups: blank group (not plus pronase), DMSO (pronase 1:1000), ATG100 μM
(pronase 1:1000), ATG300 μM (pronase 1:1000), ATG1000 μM (pronase 1:1000).Knot
In the condition of pronase 1:1000, (pronase can be with digestible protein, but it is digested not in conjunction with ATG for fruit discovery
Albumen is more than albumen of the digestion in conjunction with ATG.We test the pronase of various concentration, as a result, it has been found that 1:1000
Concentration us can be helped to find in conjunction with ATG well and not with the protein-bonded difference of ATG, it is this to be used to study small point
The protein-bonded method of sub- compound is DARTS.It can be found that PP2A is combined and is consequently increased with the increase of ATG concentration,
As a result see Fig. 2.
Fig. 2: DARTS-western blot confirms ATG in conjunction with PP2AB, and PP2A has 3 structural units, what ATG was combined
It is catalyst elements.The albumen of this catalyst elements is PP2AB, and gene is PPP2CB.
If necessary to extract PP2A albumen, then pronase is not added, using following steps:
Step 1,100 μM~1000 μM of ATG, which is added, stimulates cell.
Step 2 is extracted cell and is crushed；
Step 3, loading simultaneously carry out polyacrylamide gel electrophoresis；
Step 4 cuts the gel-tape of part corresponding with PP2A molecular weight, recycles albumen.When recycling albumen, it will coagulate
Adhesive tape band is pulverized with grinding rod, and the buffer less than 500 μ l/ block glue is wherein being added, and stands 10 hours at 4 degrees Celsius, then
5000-10000rpm is centrifuged 10min, contains PP2A albumen in supernatant.The protein recovery adopted this method is 70%.
Cell in step can be 293T cell, be also possible to the cell of other types.
3, ATG is on the active influence of PP2A
Be respectively 0,1.0 μm with concentration, 3.3 μm, 10 μm of ATG stimulate 293T cell, stimulation time is 1 hour, is utilized
PP2A active agent box (R&D systems, DYC3309-2) detects PP2A activity, as a result, it has been found that as ATG concentration increases,
The activity of PP2A is consequently increased, and the results are shown in Table 1.It can be seen that ATG can effectively increase the activity of PP2A.With the increase of ATG dosage,
The activity of PP2A is consequently increased.
Table 1:PP2A activity value
| DMSO | 1.0μm | 3.3μm | 10μm | |
| Mean ± standard deviation | 0.0176±0.0009 | 0.0268±0.0004 | 0.0279±0.0030 | 0.0484±0.0036 |
| P value | 0.006 | 0.044 | 0.007 |
Note: P value is compared with DMSO.
Claims (2)
1. application of the arctigenin in recycling PP2A albumen, which comprises the steps of:
Step 1,100 μM~1000 μM of arctigenin, which is added, stimulates 293T cell；.
Step 2 is extracted cell and is crushed；
Step 3, loading simultaneously carry out polyacrylamide gel electrophoresis；
Step 4 cuts the gel-tape of part corresponding with PP2A molecular weight, recycles albumen.
2. application of the arctigenin as claimed in claim 1 in recycling PP2A albumen, it is characterised in that:
Wherein, when recycling albumen, gel-tape is pulverized with grinding rod, the buffer less than 500 μ l/ block glue is wherein being added,
10 hours are stood at 4 degrees Celsius, then 5000-10000rpm is centrifuged 10min, contains PP2A albumen in supernatant.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610278529.9A CN105832719B (en) | 2016-04-28 | 2016-04-28 | Arctigenin is adjusting the application in PP2A activity |
| US15/461,494 US9994533B2 (en) | 2016-04-28 | 2017-03-17 | Method of preparing ATG's analogue, and the application of ATG and it's analogue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610278529.9A CN105832719B (en) | 2016-04-28 | 2016-04-28 | Arctigenin is adjusting the application in PP2A activity |
Publications (2)
| Publication Number | Publication Date |
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| CN105832719A CN105832719A (en) | 2016-08-10 |
| CN105832719B true CN105832719B (en) | 2019-05-03 |
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| CN201610278529.9A Active CN105832719B (en) | 2016-04-28 | 2016-04-28 | Arctigenin is adjusting the application in PP2A activity |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102805743A (en) * | 2011-06-03 | 2012-12-05 | 鲁南制药集团股份有限公司 | Application of arctigenin in treating angiogenesis diseases |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2832355A4 (en) * | 2012-03-26 | 2015-12-23 | Kracie Pharma Ltd | Anticancer agent |
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- 2016-04-28 CN CN201610278529.9A patent/CN105832719B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102805743A (en) * | 2011-06-03 | 2012-12-05 | 鲁南制药集团股份有限公司 | Application of arctigenin in treating angiogenesis diseases |
Non-Patent Citations (1)
| Title |
|---|
| Arctigenin (ATG) Attenuates Podocyte Injury in Diabetic Kidney through Inhibition of PP2A/NFKB/NOX4 Pathway;Yifei Zhong等;《Nephron》;20160406;289 * |
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