CN112755027A - Application of hypaconitine and structural analogs thereof as Sirt3 inhibitor and in-vivo and in-vitro oxidative stress induction - Google Patents
Application of hypaconitine and structural analogs thereof as Sirt3 inhibitor and in-vivo and in-vitro oxidative stress induction Download PDFInfo
- Publication number
- CN112755027A CN112755027A CN202110102903.0A CN202110102903A CN112755027A CN 112755027 A CN112755027 A CN 112755027A CN 202110102903 A CN202110102903 A CN 202110102903A CN 112755027 A CN112755027 A CN 112755027A
- Authority
- CN
- China
- Prior art keywords
- sirt3
- hypaconitine
- application
- oxidative stress
- vivo
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
Landscapes
- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to a new application of aconitine as a Sirt3 inhibitor, and particularly discloses an application of aconitine and a structural analogue thereof as a Sirt3 inhibitor and inducing in vivo and in vitro oxidative stress, an influence of the aconitine on the expression of cell Sirt3 protein, an application of the aconitine as a Sirt3 inhibitor, an application of the structural analogue of the aconitine as a Sirt3 inhibitor, an application of the aconitine for inducing in vivo and in vitro oxidative stress by inhibiting Sirt3, and an application of the structural analogue of the aconitine for inducing in vivo and in vitro oxidative stress by inhibiting Sirt 3. The hypaconitine has obvious inhibition effect on Sirt3, causes the increase of oxidative stress level, and can be used as the inhibitor of Sirt3 for scientific research and pharmaceutical clinic.
Description
Technical Field
The invention belongs to new application of hypaconitine and analogues thereof, and particularly relates to application of hypaconitine and analogues thereof as a Sirt3 inhibitor and for inducing oxidative stress in vivo and in vitro.
Background
NAD-dependent deacetylase Sirt3, which is a deacetylase, is mainly localized to mitochondria, Sirt3 is capable of deacetylating and activating various antioxidant enzymes (including manganese superoxide dismutase MnSOD, catalase, superoxide dismutase SOD2, glutathione reductase GSH, etc.), and thus participates in oxidative stress regulation and active oxygen level removal.
The hypaconitine diester alkaloids are present in Aconitum carmichaeli Debx, Aconitum brachypearum Debx, Aconitum coreanum (Levl.) Druce root tuber, hypaconine, and homoaconitine. It is reported that hypaconitine can directly inhibit the production of inflammatory cytokines and inflammatory mediators through multiple targets, and exert an anti-inflammatory function. Whether the aconitine can induce in vivo and in vitro oxidative stress or not is not reported, and Sirt3 expression is inhibited. .
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides application of the hypaconitine as the Sirt3 inhibitor, also provides application of a structural analogue of the hypaconitine as the Sirt3 inhibitor, also provides application of the hypaconitine in inducing in-vivo and in-vitro oxidative stress by inhibiting Sirt3, and also provides application of the structural analogue of the hypaconitine in inducing in-vivo and in-vitro oxidative stress by inhibiting Sirt 3.
Use of hypaconitine according to an embodiment of the first aspect of the present invention as an inhibitor of Sirt3, said hypaconitine having the chemical structure:
use of a structural analogue of hypaconitine according to an embodiment of the second aspect of the present invention as an inhibitor of Sirt 3.
Use of hypaconitine according to the third aspect of the present invention to induce oxidative stress in vitro and in vivo by inhibiting Sirt 3.
Use of a structural analogue of hypaconitine according to the fourth embodiment of the present invention to induce oxidative stress in vivo and in vitro by inhibiting Sirt 3.
According to the application of the aconitine and the analogue thereof as the Sirt3 inhibitor, the expression condition of the Sirt3 protein in the aconitine treated cells is detected by a western blot method, and the result shows that the aconitine has obvious inhibition effect on the Sirt3 protein.
Drawings
FIG. 1 is the chemical structure of hypaconitine;
FIG. 2 shows the detection of Sirt3 protein expression levels in cells by Western blotting;
p <0.05 in figure 3 compared to control; p <0.01(n ═ 4, mean ± SD).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The use of hypaconitine and its analogs as Sirt3 inhibitors according to embodiments of the present invention is described below with reference to figures 1-3.
According to the application of the hypaconitine and the analogue thereof as the Sirt3 inhibitor, the chemical structure of the hypaconitine is as follows:
wherein, the application of the hypaconitine and the structural analogue thereof in inducing in-vivo and in-vitro oxidative stress by inhibiting Sirt 3.
Aconitine acts as an inhibitor of Sirt3, and the effect of aconitine on Sirt3 protein expression was examined using a Western immunoblotting assay.
(1) Cell culture: the H9c2 cardiomyocytes were cultured in high-glucose DMEM containing 10% fetal bovine serum and the cells were cultured in a 5% CO2 incubator at 37 ℃. When the culture dish is filled with the myocardial cells by about 90 percent, the culture solution is discarded, the cells are soaked and washed by PBS for three times, then the PBS is discarded, 0.25 percent of pancreatin is added to digest the cells, the cells are observed under a microscope, when the cells retract and become round, the PBS is rapidly added to stop the digestion, the myocardial cells are blown off by a pipette, then the cell suspension is transferred to a 4mL centrifuge tube and centrifuged for 2 minutes under the condition of 800 rpm. The upper layer of culture medium was discarded, and the cells were resuspended in 1ml of fresh culture medium and cultured in 6-well plates as needed. After 24 hours, the cardiomyocytes H9c2 appeared in the form of long spindle when observed under an inverted microscope, and well-growing cells were used for the experiment.
(2) Cell protein extraction
Washing cells with PBS, adding 200 μ L of protein lysate into each well of 6-well plate, and placing on a horizontal shaker to shake vigorously for 2 min; blowing down the cells by using a pipette, transferring the cells into a 1.5mL centrifuge tube, shaking the cells by using a vortex oscillator, standing the cells on ice for 5min, repeating the process for 1 time, finally shaking the cells again, placing the cells in a 4C centrifuge, centrifuging the cells at the highest speed for 10min, transferring the supernatant into a new 1.5mL centrifuge tube, and storing the centrifuge tube in a refrigerator at the temperature of-20 ℃ for later use.
(3) BCA method for determining protein concentration
Reagent preparation
Reagent A: BCA 1g, Na2CO31.6958 g, sodium tartrate 0.161g, NaHCO30.9241g and NaOH 0.4 g. The volume was made up to 100ml with dH 2O.
Reagent B: 0.4g of CuSO4.5H2O was dissolved in 10ml of dH 2O.
The protein standard substance is BSA with the concentration of 0.1-0.5 mg/ml.
Mixing a BCA working solution: a, B is 50: 1.
③ adding 20 mul BSA standard or diluted sample into 250 mul BCA, mixing evenly, reacting for 30min at 60 ℃.
Cooling. The absorbance at 562nm was measured and the sample concentration was calculated.
(4)Western blot
1) Glue preparation
Separating gel and concentrated gel are prepared according to the formula shown in the specification.
2) Preparation of protein samples
And (4) calculating the sample loading volume of the protein sample according to the concentration measured in the step (4), supplementing each sample to a uniform volume by using a protein lysate, adding a 5 x loading buffer, shaking, uniformly mixing and centrifuging, placing on a metal bath at 100 ℃ for boiling for 5min, shaking, uniformly mixing after centrifuging, centrifuging again, depositing liquid in the tube to the end of the tube electrophoresis, taking down the separation gel, and performing the following steps.
3) Gel electrophoresis
Installing an electrophoresis device, adding a 1 Xelectrophoresis buffer solution precooled at 4 ℃ into an electrophoresis tank, adding the protein sample obtained in the previous step into a concentrated gel hole by using a liquid transfer machine, and carrying out electrophoresis for 90 minutes at a constant voltage of 124V.
4) The film transferring book is provided with a film transferring device and is arranged on a magnetic stirrer, and the film is transferred for 1h under the constant pressure of 100V.
5) Sealing of
Shearing an NC membrane into a proper size according to a protein Maker, soaking the NC membrane in 5% skimmed milk, placing the skimmed milk on a horizontal shaking table, and slowly shaking and sealing for 1h at room temperature.
6) Antibody incubation
Sucking off milk, adding corresponding primary antibody, and placing on a shaking table in a refrigerator at 4 ℃ to slowly shake and incubate overnight; after incubation, primary antibody was recovered, and the membrane was washed 3 times with 1 × PBST containing 0.5% tween 20, 8min each time; after washing, the secondary antibody is added, placed on a horizontal shaking table and slowly shaken at room temperature. Incubating for 1 h; the secondary antibody was recovered and the membrane washed 3 times with 1 XPBST for 8min each.
7) ECL detection
And (3) uniformly mixing the ECLA solution and the B solution according to the proportion of 1:1, dripping the mixture onto an NC membrane, and photographing for imaging.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (4)
2. use of structural analogs of hypaconitine as Sirt3 inhibitors.
3. Use of hypaconitine for inducing in vivo and in vitro oxidative stress by inhibiting Sirt3 is provided.
4. The structural analogue of hypaconitine induces in vivo and in vitro oxidative stress by inhibiting Sirt 3.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110102903.0A CN112755027A (en) | 2021-01-26 | 2021-01-26 | Application of hypaconitine and structural analogs thereof as Sirt3 inhibitor and in-vivo and in-vitro oxidative stress induction |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110102903.0A CN112755027A (en) | 2021-01-26 | 2021-01-26 | Application of hypaconitine and structural analogs thereof as Sirt3 inhibitor and in-vivo and in-vitro oxidative stress induction |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112755027A true CN112755027A (en) | 2021-05-07 |
Family
ID=75705740
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110102903.0A Pending CN112755027A (en) | 2021-01-26 | 2021-01-26 | Application of hypaconitine and structural analogs thereof as Sirt3 inhibitor and in-vivo and in-vitro oxidative stress induction |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112755027A (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107073054A (en) * | 2014-08-01 | 2017-08-18 | 株式会社资生堂 | Longevity gene expression facilitator |
-
2021
- 2021-01-26 CN CN202110102903.0A patent/CN112755027A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107073054A (en) * | 2014-08-01 | 2017-08-18 | 株式会社资生堂 | Longevity gene expression facilitator |
Non-Patent Citations (5)
Title |
---|
WANG NING NING 等: "Aconitine attenuates mitochondrial dysfunction of cardiomyocytes via promoting deacetylation of cyclophilin-D mediated by sirtuin-3", 《JOURNAL OF ETHNOPHARMACOLOGY》 * |
方堃 等: "次乌头碱对H2O2致大鼠心肌细胞凋亡的保护作用", 《中国中医药科技》 * |
李志勇: "附子成分次乌头碱心脏毒性及中毒机制研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
潘校琦 等: "中药药源性心脏毒性研究进展", 《药学进展》 * |
陈贝贝: "SIRT3与氧化应激及其与运动关系的研究进展", 《体育科技》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Rodrigues et al. | Dissolvable microcarriers allow scalable expansion and harvesting of human induced pluripotent stem cells under xeno‐free conditions | |
Sottocasa et al. | An electron-transport system associated with the outer membrane of liver mitochondria: a biochemical and morphological study | |
Kijimoto et al. | Enhanced glycolipid: α-galactosyltransferase activity in contact-inhibited hamster cells, and loss of this response in polyoma transformants | |
CN106635976B (en) | Method and kit for obtaining amniotic mesenchymal stem cells | |
US20180328927A1 (en) | Methionine adenosyltransferase (mat) biological activity assay and detection kit | |
EP0277837A3 (en) | Methods for separating malignant cells from clinical specimens | |
CN112755027A (en) | Application of hypaconitine and structural analogs thereof as Sirt3 inhibitor and in-vivo and in-vitro oxidative stress induction | |
CN106867964B (en) | Myocardial cell culture solution and culture method | |
Antonowicz et al. | Cystic fibrosis: lysosomal and mitochondrial enzyme activities of lymphoid cell lines | |
Narahara et al. | Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media | |
CN111172104A (en) | Separation culture method of umbilical blood mesenchymal stem cells | |
CN110885784B (en) | Clinical application-grade adipose-derived stem cells and preparation method thereof | |
CN113292546B (en) | Mangiferin derivative and preparation method and application thereof | |
CN115678841A (en) | Composition for enhancing immune suppression function of human umbilical cord mesenchymal stem cells and application | |
CN110592007B (en) | Mesenchymal stem cell and preparation method and application thereof | |
US20110143390A1 (en) | Method for testing drug sensitivity and device used therefor | |
CN114295837A (en) | Erythropoietin detection method based on magnetic beads as carriers | |
CN106867965B (en) | Efficient induction differentiation agent and induction differentiation culture medium for adipose-derived stem cells | |
CN112980006A (en) | Protein cross-linked nano affinity microsphere, preparation method and application | |
Imagawa et al. | Collagen gel method for the primary culture of mouse mammary epithelium | |
CN114366729A (en) | Application of U0126 as ADH agonist | |
CN111394301A (en) | Application of piceatannol in increasing number of secreted exosomes of pluripotent stem cells and bioactivity | |
Thacore et al. | Isolation and characterization of lysosomerich fractions from cultured cells | |
CN114621919B (en) | Method for enhancing immunoregulation capability of mesenchymal stem cells | |
CN114561362B (en) | Separation method of human-derived kidney syndrome hemorrhagic fever Hantaan virus |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |