CN114917236A - Application of tanshinone in preparation of GPX4 agonist - Google Patents

Abstract

The invention relates to an application of tanshinone in preparing GPX4 agonist, and relates to the field of medicine. The tanshinone effectively promotes the expression of GPX4 in the myocardial cells, thereby inhibiting the myocardial cell iron death, and can be used for developing a medicament for treating and/or preventing the myocardial cell iron death.

Description

Application of tanshinone in preparation of GPX4 agonist

Technical Field

The invention relates to the field of medicines, in particular to application of tanshinone in preparation of GPX4 agonist.

Background

Myocardial infarction is a common critical clinical condition. After AMI occurs, even if the blood circulation is successfully reestablished in early stage, the myocardial cell can only save ischemia and suppress myocardium, and the myocardial cell, as a terminally differentiated cell, once dying, can cause permanent loss of heart function unit, and then cause a series of changes of heart structure and function, finally cause occurrence and development of various cardiovascular diseases including malignant arrhythmia, heart failure and sudden cardiac death. Researches find that the death of the myocardial cells is a common pathogenic basis for the occurrence and development of various cardiovascular diseases, and how to effectively inhibit the death of the myocardial cells after myocardial infarction and protect the heart function is still a difficult problem to be solved urgently in the field of cardiovascular disease researches.

Iron death (Ferroptosis) is an iron-dependent non-apoptotic cell death mode newly discovered in recent years, and is mainly characterized by iron-dependent lipid active oxygen increase and is closely related to iron metabolism abnormality, lipid peroxidation and the like. In recent years, it has been found that the heart can cause excessive accumulation of iron, accumulation of active oxygen and pathological conversion of membrane lipids in the event of myocardial ischemia, etc., which constitute important factors of iron death, and can exhibit characteristics related to iron death, such as iron overload, oxidative stress, endoplasmic reticulum stress and mitochondrial dysfunction. The application of iron death inhibitor or iron chelator can improve cardiac function, and prevent and treat myocardial injury and heart failure induced by iron death.

Among the known regulatory genes for preventing iron death, Glutathione peroxidase 4 (GPX 4) is an important antioxidant enzyme that uses Glutathione (GSH) as a substrate to reduce and prevent the accumulation of lipid Reactive Oxygen Species (ROS) and thus protect cells from iron death caused by oxidative stress and lipid peroxidation. GSH consists of glutamic acid (Glutamate), Cysteine (Cysteine), and Glycine (Glycine). It has been found that inhibition of GSH synthesis (e.g., Erastin iron death inducer) by preventing cysteine uptake results in the formation and accumulation of iron-dependent lipid peroxidation products. Further research on the action mechanism shows that, for example, Erastin reduces GSH synthesis mainly by inhibiting cysteine intake through inhibiting cystine/glutamate antiporter System (System Xc-), GSH exhaustion causes GPX4 activity to be reduced, lipid peroxide cannot be reduced, and then divalent iron ions oxidize lipid to generate a large amount of active oxygen, thereby causing accumulation of toxic peroxide, damage to protein and cell membrane and subsequent cell iron death. A plurality of previous researches also prove that the depletion of GPX4 has the effect of intensifying the sensitivity of myocardial cells to iron death, and the promotion of the expression of GPX4 has the effect of inhibiting the iron death of the myocardial cells. In addition, Tae-Jun Park et al also revealed by proteomic analysis that iron death during MI was also involved in mediating cardiomyocyte death and cardiac injury, and its mechanism of action was probably related to the decreased expression of GPX 4. This further demonstrates that GPX4 plays an important role in mediating cardiomyocyte death.

In conclusion, as an important form of cardiomyocyte death, GPX4 can be used as a therapeutic target for preventing and treating cardiomyocyte iron death. However, there is currently a lack of effective agents for inhibiting myocardial cell iron death.

Disclosure of Invention

Aiming at the problems, the invention provides application of tanshinone in preparing GPX4 agonist, which can inhibit myocardial cell iron death by effectively promoting expression of GPX4 in myocardial cells, and can be used for developing medicines for treating and/or preventing myocardial cell iron death.

In order to achieve the purpose, the invention provides application of tanshinone in preparing GPX4 agonist.

In the research process, the inventor finds that tanshinone has the effect of promoting GPX4 expression and inhibiting myocardial cell iron death.

The invention also provides application of tanshinone in preparing a medicament for preventing and/or treating myocardial cell iron death.

In one embodiment, the tanshinone plays a role in preventing and/or treating myocardial cell iron death by promoting expression of GPX4, reducing contents of ferrous iron, ROS and MDA in myocardial cells and improving content of GSH and activity of SOD.

The invention also provides a method for inhibiting the death of the myocardial cell iron, which adopts tanshinone to intervene in the treatment of the myocardial cell.

The invention also provides a method for inhibiting myocardial cell iron death for scientific research purposes, which comprises the following steps: the tanshinone is used for intervening and treating the myocardial cells.

In one embodiment, the intervention process comprises the steps of: contacting myocardial cells with tanshinone.

In one embodiment, the concentration of tanshinone is 5 μ g/mL-50 μ g/mL.

The tanshinone with the working concentration can promote the expression of GPX4 and inhibit the death of myocardial cells.

In one embodiment, the concentration of tanshinone is 8 μ g/mL to 45 μ g/mL.

In one embodiment, the concentration of tanshinone is 35 μ g/mL-45 μ g/mL.

In one embodiment, the concentration of tanshinone is 8 μ g/mL-12 μ g/mL.

In one embodiment, the concentration of tanshinone is 18 μ g/mL-22 μ g/mL.

Compared with the prior art, the invention has the following beneficial effects:

the application of tanshinone in preparing GPX4 agonist promotes the expression of GPX4 through high-efficiency and low-toxicity tanshinone, protects myocardial cells from iron death caused by lipid peroxidation, has low cost, can be used for developing a GPX4 agonist with a promising prospect, and has an excellent application prospect in the development of drugs for preventing and treating myocardial cell iron death.

Drawings

FIG. 1 is a graph showing the results of measuring the effect of tanshinone on the ferrous ion content of cardiomyocytes in example 3;

FIG. 2 is a graph showing the results of measuring the effect of tanshinone on the ROS content of cardiomyocytes in example 4;

FIG. 3 is a graph showing the results of measurement of the effect of tanshinone on the amount of MDA in myocardial cells in example 5;

FIG. 4 is a graph showing the measurement results of the effect of tanshinone on the content of GSH in cardiomyocytes in example 6;

FIG. 5 is a graph showing the results of measuring the effects of tanshinone on the SOD activity of cardiomyocytes in example 7;

fig. 6 is a graph of the results of measuring the effect of tanshinone on GPX4 protein expression in cardiomyocytes in example 8, where a is a graph of the results of Western blot detection of GPX4 expression in iron-dead cardiomyocytes, and B is a graph of the results of measurement of the relative expression of GPX 4.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Defining:

the tanshinone: is liposoluble phenanthrenequinone compound extracted from root and stem of Salvia miltiorrhiza Bge.

The source is as follows:

rat myocardial cell H9C2 (purchased from cell bank of Chinese academy of sciences), cell MDA detection kit (Nanjing institute of bioengineering, Cat: A003-4-1), GSH detection kit (Nanjing institute of bioengineering, Cat: A006-2-1), ROS detection kit (Shanghai Biyutian Biotechnology Co., Ltd., Cat: S0033S), SOD detection kit (Shanghai Biyutian Biotechnology Co., Ltd., Cat: S0101M), cell ferrous content detection kit (Beijing Edison Biotechnology Co., Ltd.), Rabbitani-GPX 4(ab125066, Abcam), Rabbitani-GAPDH AF7021, Affinity), Erastin (MCHY 15763, MCHY), Feostatin-1 (MCRRE-100579, tanshinone), and tanshinone (batch MUST 20010801).

Reagents, materials and equipment used in the present example are all commercially available sources unless otherwise specified; unless otherwise specified, all the methods are conventional in the art.

Example 1

Construct in vitro iron-dead cell model.

Rat cardiomyocytes H9C2 were cultured in cell incubator (95% air and 5% CO) using high-glucose DMEM medium ₂ ) As Con group.

Then an iron death inducer Erastin (10 mu M) construct external iron death cell model is adopted as an Erastin group, and the construction method of the external iron death cell model comprises the following steps: firstly, 1mg of Erastin is dissolved in 182.8 mul of DMSO solution to ensure that the final concentration is 10mM, and when rat myocardial cells in a cell culture dish grow and fuse to about 90%, the mass ratio of Erastin: cell culture medium ═ 1: 1000 into the cell culture medium to make the Erastin final concentration 10. mu.M.

Example 2

And preparing the reagent.

1. Preparing a tanshinone reagent.

Tanshinone is respectively prepared into a tanshinone reagent 1 (the concentration is 10mg/mL), a tanshinone reagent 2 (the concentration is 20mg/mL) and a tanshinone reagent 3 (the concentration is 40mg/mL) by adopting DMSO.

Treating cells by using the tanshinone reagent according to the proportion of 1: 1000 to obtain a sample 1 to be tested (the working concentration of tanshinone is 10 mug/mL), a sample 2 to be tested (the working concentration of tanshinone is 20 mug/mL) and a sample 3 to be tested (the working concentration of tanshinone is 40 mug/mL) by adding the working concentrations of tanshinone to the Erastin group cell culture medium prepared in example 1 to make the working concentrations of tanshinone be 10 mug/mL, 20 mug/mL and 40 mug/mL.

2. Preparing a Ferrostatin-1 reagent.

10mg of Fer-1 was dissolved in 381.17 μ LDMSO according to the molecular weight (MW: 262.35) of Ferrostin-1 (Fer-1), prepared to a concentration of 100mM stock solution, and stored at-80 ℃ for use.

Cells were treated with the above described Ferrostatin-1 reagent at a rate of 1: 1000, and adding the mixture into the Erastin cell culture medium prepared in example 1 to make the working concentration of the Ferrostatin-1 reagent 10 μ M, so as to obtain a sample 4 to be detected (the working concentration of the Ferrostatin-1 reagent is 10 μ M).

In example 1, the Con group is sample 5 to be tested, and the Erastin group is sample 6 to be tested (working concentration of Erastin is 10 μ M).

Example 3

And (5) detecting the ferrous content of the cells.

The detection method comprises the following steps: the sample to be tested in example 2 was collected in a grinding tube using the extract according to the instructions of the cellular ferrous kit (A-DS-WQT207), ground using a homogenizer, centrifuged at 12000rpm at 4 ℃ for 10min, and the supernatant was collected and placed on ice. The solutions in each set of test tubes were then prepared in the amounts shown in the following table.

TABLE 1 amount of reagents used in each set of tubes in the detection of ferrous content in cells

The 8 groups of detection tubes were mixed well, left to stand at room temperature for 15min, and centrifuged at 3000rpm for 5min if turbid, 200. mu.L of supernatant was put into a 96-well plate, and the absorbance of each tube was read at 562nm, the results are shown in FIG. 1.

Example 4

And (4) detecting ROS of the cells.

The detection method comprises the following steps: according to ROS kit instructions, as per 1: 1000 ratio with serum-free medium dilution DCFH-DA, to make DCFH-DA working concentration of 10 u mol/L.

An appropriate volume of diluted DCFH-DA (the volume added is appropriate to cover the cells sufficiently) was added to the test sample of example 2. Incubate at 37 ℃ for 20 minutes in a cell incubator. Cells were washed three times with serum-free cell culture medium to remove DCFH-DA well without entering the cells. And detecting by using a fluorescence microplate reader. The results are shown in FIG. 2.

Example 5

And (5) detecting cell MDA.

The detection method comprises the following steps: according to the instructions of the cell MDA detection kit (A003-4-1), the sample to be tested in example 2 was collected in a grinding tube using the extracting solution, and after grinding the sample with a homogenizer, 0.1mL of the sample was put in a 1.5mL centrifuge tube (a small hole was punched on the tube cover in advance), and then solutions in the detection tubes of each group were prepared in the amounts shown in the following table.

TABLE 2 amount of reagents used in each set of test tubes in the cell MDA test

The 8 groups of detection tubes are covered with a cover, fully and uniformly mixed, water bath at 95 ℃ is carried out for 40min, the detection tubes are taken out and cooled by running water, centrifugation is carried out at 4000rpm for 10min, 0.25mL of reaction liquid of each tube is absorbed and added into a 96-well plate, and the absorbance of each hole is measured at the wavelength of 530nm of an enzyme-labeling instrument, and the result is shown in figure 3.

Example 6

And (5) detecting the cell GSH by using a kit.

The detection method comprises the following steps: according to the specification of the GSH kit, collecting the sample to be detected in example 2 in a grinding tube, fully homogenizing, adding 0.1mL of the homogenized sample into 0.1mL of reagent I of the cell GSH kit, fully and uniformly mixing, 3500rpm, centrifuging for 10min, taking supernate, and preparing the solution in each group of detection holes according to the dosage in the following table.

TABLE 3 amount of reagents used in each set of test tubes in the assay of the cellular GSH kit

Mixing the above 8 groups of detection holes, standing for 5min at 405nm, and measuring absorbance value of the separation hole with enzyme labeling instrument, the result is shown in FIG. 4.

Example 7

And (5) detecting the cell SOD kit.

The detection method comprises the following steps: according to the specification of the SOD detection kit: the test sample of example 2 was appropriately blown with the SOD sample preparation solution to sufficiently lyse the cells. Centrifuging at 12000rpm for 3-5 min at 4 deg.C, and collecting supernatant for detection.

Preparation of WST-8/enzyme working solution: WST-8/enzyme working solutions for the samples to be tested (6 groups) and for the blank (3 groups) of example 2 were prepared according to the following table.

TABLE 4 reagent dosage of WST-8/enzyme working solution in cell SOD kit detection

Detecting the amount of sample	9
		SOD detection buffer solution (mu L)	1359
WST-8(μL)	72
		Enzyme solution (μ L)	9
WST-8/enzyme working solution (uL)	1440

Preparing a reaction starting working solution: and (3) melting the reaction starting liquid (40X) in the kit, uniformly mixing, diluting according to the proportion that 39 mu L of SOD detection buffer solution is added into every 1 mu L of reaction starting liquid (40X), and uniformly mixing to obtain the reaction starting working solution. The prepared reaction starting working solution is stored at 4 ℃ and is ready to use.

The solutions in each set of test tubes were then prepared according to the amounts used in the cell SOD kit test in the following table, and the results are shown in FIG. 5.

TABLE 5 dosage of reagents in each group of detection tubes in cell SOD kit detection

Example 8

And detecting Western blot.

The detection method comprises the following steps: according to the protease inhibitors: phosphatase inhibitors: RIPA cell lysate ═ 1: 1: 100, adding an appropriate amount of lysate to the sample to be tested in example 2, fully lysing the cells, scraping the cells with a cell scraper, collecting the cells into a 1.5ml EP tube, performing the whole process on ice at 4 ℃, 12000rpm, centrifuging for 15 minutes, transferring the supernatant into another 1.5ml EP tube, performing protein quantification, performing denaturation, performing electrophoresis, performing membrane transfer, incubating anti-GPX4 (1: 10000), performing GAPDH (1: 1000) overnight in a refrigerator with a shaking table at 4 ℃, performing secondary antibody at room temperature for 1h, finally performing exposure, and analyzing the result, wherein the result is shown in fig. 6.

Example 9

And (6) analyzing the result.

The invention adopts an Erastin (10 mu M) construct external iron death myocardial cell model to observe the influence of tanshinone on myocardial cell iron death and GPX4 expression.

As shown in figure 1, compared with the control group, the ferrous content in the iron death cardiac muscle cell model is obviously increased (P is less than 0.01), and tanshinone has the dose-dependent effect of reducing the ferrous content (P is less than 0.01), and the effect of the tanshinone is consistent with the effect of the iron death inhibitor Fer-1 (10 mu M).

Further detecting ROS, MDA, GSH and SOD, finding out that the results are shown in figures 2, 3, 4 and 5, compared with a control group, the contents of ROS and MDA in the iron-dead myocardial cell model are both obviously increased (P is less than 0.01), and the contents of GSH and SOD activity are both obviously reduced (P is less than 0.01); tanshinone has effects of reducing ROS and MDA content (P is less than 0.01); increase GSH content and SOD activity (P is less than 0.01), and the effect is consistent with that of Fer-1.

Further, Western-blot is adopted to detect the expression of GPX4, and the result is shown in A and B of FIG. 6, the expression of GPX4 in an Erastin-induced iron-killed cardiomyocyte model is remarkably reduced (P is less than 0.01), and tanshinone has the effect of promoting the expression of GPX4 (P is less than 0.01), and the effect of tanshinone is consistent with the effect of Fer-1.

In conclusion, tanshinone has the effect of inhibiting myocardial cell iron death by promoting the expression of GPX 4.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (9)

1. Application of tanshinone in preparing GPX4 agonist is provided.

2. Application of tanshinone in preparing medicine for preventing and/or treating myocardial cell iron death is provided.

3. The use as claimed in claim 2, wherein the tanshinone has effects of preventing and/or treating myocardial cell iron death by promoting expression of GPX4, reducing content of ferrous iron, ROS and MDA in myocardial cell, and increasing content of GSH and SOD activity.

4. A method for inhibiting myocardial cell iron death is characterized in that tanshinone is used for intervening and treating myocardial cells.

5. The method as recited in claim 4, wherein the concentration of tanshinone is 5 μ g/mL-50 μ g/mL.

6. The method as recited in claim 5, wherein the concentration of tanshinone is 8 μ g/mL-45 μ g/mL.

7. The method as recited in claim 6, wherein the concentration of tanshinone is 35 μ g/mL-45 μ g/mL.

8. The method as recited in claim 6, wherein the concentration of tanshinone is 8 μ g/mL-12 μ g/mL.

9. The method as set forth in claim 6, wherein the concentration of tanshinone is 18 μ g/mL to 22 μ g/mL.

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