阅读说明：本技术 次乌头碱及其结构类似物作为Sirt3抑制剂和诱导体内外氧化应激的应用 (Application of hypaconitine and structural analogs thereof as Sirt3 inhibitor and in-vivo and in-vitro oxidative stress induction ) 是由 段亚君 黄蓉 徐帅 王垣钰 韩际宏 张爽 杨潇潇 陈元利 于 2021-01-26 设计创作，主要内容包括：本发明属于次乌头碱作为Sirt3抑制剂的新用途,具体公开了次乌头碱及其结构类似物作为Sirt3抑制剂和诱导体内外氧化应激的应用,次乌头碱对细胞Sirt3蛋白表达的影响,次乌头碱作为Sirt3抑制剂的应用,次乌头碱的结构类似物作为Sirt3抑制剂的应用,次乌头碱通过抑制Sirt3诱导体内外氧化应激的应用,次乌头碱的结构类似物通过抑制Sirt3诱导体内外氧化应激的应用。次乌头碱对Sirt3有明显的抑制作用,并且导致氧化应激水平升高,可以成为Sirt3的抑制剂用于科学研究以及药物临床。(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.)

1. The application of the hypaconitine as the Sirt3 inhibitor is characterized in that the chemical structure of the hypaconitine is as follows:

。

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.

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.