Application of caffeol palmitate, GPX4 binding preparation and PML inhibitor

Abstract

The embodiment of the invention discloses application of caffeol palmitate, a GPX4 binding preparation and a PML inhibitor. The application of the caffeol palmitate can be expanded.

Description

TECHNICAL FIELD

[0001] The present application relates to the application technical field of cafestol palmitate, in particular to the use of cafestol palmitate, GPX4 binding preparation and PML inhibitor. BACKGROUND

[0002] Cafestol palmitate (CP) is an active substance derived from coffee beans, and its chemical structural formula is shown in Figure 1 , and its CAS number is 81760-46-5.

[0003] At present, it has been found that the main uses of cafestol palmitate include treating diabetes (see patent literature with patent number

[0004] EP3148525B1), and enhancing lipid barrier synthesis (see patent literature with patent number US6716437B1). However, in addition to the above, the application of cafestol palmitate is still less.

[0005] For example, how to use cafestol palmitate to regulate glutathione peroxidase 4 (GPX4) or promyelocytic leukemia protein (PML) is rarely reported.

[0006] Therefore, in general, the research on cafestol palmitate in the prior art is less. SUMMARY

[0007] One purpose of the embodiments of the present application is to solve at least one problem in the above background, and to provide corresponding beneficial effects.

[0008] Another purpose of the embodiments of the present application is to provide a use of cafestol palmitate, a GPX4 binding preparation and an inhibitor, which can expand the new application of cafestol palmitate.

[0009] The embodiments of the present application mainly achieve the above purposes through the following technical solutions.

[0010] In a first aspect, the embodiments of the present application provide a use of cafestol palmitate in preparing a GPX4 binding preparation.

[0011] It should be noted that GPX4 refers to glutathione peroxidase 4.

[0012] In some technical solutions, according to the use of the first aspect, the GPX4 binding preparation is used to activate the activity of GPX4.

[0013] In some embodiments, the use according to the first aspect, the GPX4-binding agent is for inhibiting PML protein expression.

[0014] In some embodiments, the use according to the first aspect, the GPX4-binding agent is for inhibiting formation of PML nucleosomes.

[0015] In a second aspect, the present application provides a use of a CP in the preparation of a GPX4 activity-activating agent.

[0016] In a third aspect, the present application provides a use of a CP in the preparation of a PML inhibitor.

[0017] In some embodiments, the use according to the third aspect, the PML inhibitor is for inhibiting PML protein expression,

[0018] In some embodiments, the use according to the third aspect, the PML inhibitor is for inhibiting formation of PML nucleosomes.

[0019] In some embodiments, the use according to the third aspect, the PML inhibitor is for inhibiting cell senescence.

[0020] In a fourth aspect, the present application provides a use of a CP in the preparation of a cell senescence inhibitor.

[0021] In a fifth aspect, the present application provides a GPX4-binding agent comprising a CP.

[0022] In a sixth aspect, the present application provides an inhibitor comprising a CP.

[0023] The beneficial effects of the embodiments of the present application include:

[0024] In some embodiments, the present application provides a use of a CP in the preparation of a GPX4-binding agent. Specifically, the CP has a binding relationship with GPX4, and the GPX4-binding agent can bind to GPX4 through the CP.

[0025] In some embodiments, the present application also provides a use of a CP in the preparation of a GPX4 activity-activating agent. The CP has a significant activating effect on the enzyme activity of GPX4, and thus the GPX4 activity-activating agent can activate the enzyme activity of GPX4 through the CP.

[0026] In some embodiments, the present application also provides a use of a CP in the preparation of a PML inhibitor. The PML inhibitor can reduce the level of PML protein up-regulated by RSL3 through the CP, thereby producing an inhibitory effect.

[0027] In some embodiments, the present application also provides a use of a CP in the preparation of a PML inhibitor, the PML inhibitor is capable of reducing the formation of PML nucleosomes induced by RSL3 by the CP, to produce an inhibitory effect.

[0028] In some embodiments, the present application provides a GPX4 binding agent comprising a CP. The GPX4 binding agent can be used for the enzymatic activity of GPX4, and / or for inhibiting the expression of PML protein, and / or for inhibiting the formation of PML nucleosomes, and / or for inhibiting cell senescence.

[0029] In some embodiments, the present application provides an inhibitor comprising a CP. The inhibitor is a PML inhibitor, which can be used for inhibiting the expression of PML protein, and / or for inhibiting the formation of PML nucleosomes, and / or for inhibiting cell senescence. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The chemical structure of coffee alcohol palmitate is as follows:

[0031] Figure 2 The figure is a schematic diagram of the GPX4 overexpression effect of the GPX4 overexpression HEK293 cell solution in Experimental Example 1 of the present application;

[0032] Figure 3 The figure is a schematic diagram of the GPX4 enzyme activity (GPX4 activity) of the control group and experimental groups 1-3 in Experimental Example 1 of the present application;

[0033] Figure 4 The figure is a schematic diagram of the content of GPX4 and GAPDH proteins at different temperatures of the administration group and the control group in Experimental Example 2 of the present application;

[0034] Figure 5 The figure is a schematic diagram of the dissolution curve of GPX4 protein at different temperatures of the administration group and the control group in Experimental Example 2 of the present application;

[0035] Figure 6 The figure is a schematic diagram of the dissolution curve of GAPDH protein at different temperatures of the administration group and the control group in Experimental Example 2 of the present application;

[0036] Figure 7 The figure is a schematic diagram of the binding ability of CP and GPX4 analyzed by MST in Experimental Example 3 of the present application;

[0037] Figure 8 The figure is a schematic diagram of the relative expression level of PML protein and GAPDH protein of each group in Experimental Example 4 of the present application;

[0038] Figure 9A schematic diagram of a relative gray value ratio of PML protein to GAPDH protein of each group in Experimental Example 4 of the present application is shown in Figure 1.

[0039] Figure 10 A schematic diagram of a cell fluorescence of each group in Experimental Example 5 of the present application is shown in Figure 2. DETAILED DESCRIPTION

[0040] The present application will be further described below in conjunction with the accompanying drawings, so that those skilled in the art can implement the present application according to the description in the specification.

[0041] The following terms are used in the present application, and are understood as follows unless otherwise stated.

[0042] The terms "first", "second", and the like, are used only for descriptive purposes and do not necessarily connote an order of importance, unless otherwise indicated. Thus, a feature described as "first" can imply that the feature is either explicitly or implicitly preceded by another feature, or that the feature is either explicitly or implicitly followed by another feature.

[0043] Furthermore, the terms "comprise", "comprising", "contain", "containing", "include", "including" and "have", "having" and any variations thereof are intended to cover a non-exclusive inclusion. For example, a process, method, system, product or apparatus that comprises a list of steps or elements is not necessarily limited to the listed steps or elements, but can optionally include additional steps or elements not expressly listed or inherent to such process, method, system, product or apparatus.

[0044] The term "DMSO" is the English full name of Dimethyl sulfoxide, and the Chinese name is dimethyl sulfoxide.

[0045] The term "PBS" is the English full name of phosphate buffered saline, and the Chinese full name is phosphate buffered saline. In addition, in the experimental examples provided below, the term "PBS buffer" can be used interchangeably with "PBS" in the same meaning.

[0046] The term "TBST" is the English full name of Tris-Buffered Saline with Tween-20.

[0047] In addition to the above, it is still necessary to emphasize that the reference to "embodiments" in this document means that the specific features, structures or characteristics described in connection with the embodiments can be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily mean the same embodiment, nor is it an independent or alternative embodiment to other embodiments. Those skilled in the art explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0048] <Use of CP for binding with GPX4>

[0049] Glutathione Peroxidase 4 (GPX4) is a kind of vital antioxidant enzyme in cells, and is known as the Gatekeeper of Ferroptosis. Its activity directly determines whether the cell can effectively resist ferroptosis, an iron-dependent, lipid peroxidation-driven programmed cell death. The existing technology has not found the regulation of GPX4 by coffee palmitate, which is an important research direction.

[0050] In order to provide more applications of CP in regulating GPX4, the embodiments of the present application provide a use of CP in preparing a GPX4 binding preparation.

[0051] CP has a binding effect with GPX4, and the GPX4 binding preparation can be combined with GPX4 through CP, and then can be used to regulate GPX4.

[0052] In some embodiments, the concentration of CP in the GPX4 binding preparation is 2-500 μM, optionally 50-200 μM, and optionally 50, 100 or 200 μM.

[0053] In some embodiments, the GPX4 binding preparation comprises CP and DMSO.

[0054] <Use of CP for activating GPX4>

[0055] In order to provide more applications of CP in regulating GPX4, the embodiments of the present application also provide a use of CP in preparing a GPX4 activity activating preparation.

[0056] If GPX4 is inhibited, it may cause up-regulation of PML protein and induce cell aging. CP has a significant activating effect on the enzyme activity of GPX4, and thus the GPX4 activity activating preparation can activate the enzyme activity of GPX4 through CP.

[0057] In some embodiments, the concentration of CP in the GPX4 activity activating preparation is 250-500 μM, which is more conducive to activating the enzyme activity of GPX4.

[0058] In some embodiments, the concentration of CP in the GPX4 activity activating preparation is 500 μM, which is more conducive to activating the enzyme activity of GPX4.

[0059] <Use of CP for inhibiting PML>

[0060] Promyelocytic leukemia (PML) is a subnuclear protein structure involved in a variety of important cellular functions. The most prominent feature of PML protein is the formation of structures called PML-nuclear bodies (PML-NBs) within the nucleus. These structures are not simple deposition sites, but rather highly dynamic, powerful molecular scaffolds that can recruit a large number of key proteins, greatly enhancing the efficiency of specific biochemical reactions. When PML protein is up-regulated, more and larger PML nuclear bodies are formed. These nuclear bodies become an ideal platform for assembling "senescence-associated protein complexes", thus promoting cell senescence. There is no prior art found that coffee palmitate can regulate PML, which is an important research direction.

[0061] In order to provide more applications of CP in regulating PML, the embodiments of the present application also provide a use of CP in the preparation of a PML inhibitor.

[0062] In some embodiments, the PML inhibitor can be used to inhibit the expression of PML protein. Specifically, RSL3 is an inhibitor of glutathione peroxidase 4 (GPX4), and RSL3 can induce significant up-regulation of PML protein, and CP can reverse the phenomenon of increased expression level of PML protein induced by RSL3. The PML inhibitor can reduce the level of PML protein up-regulated by RSL3 through CP, thereby producing an inhibitory effect.

[0063] RSL3-induced up-regulation of PML protein can cause cell senescence, and the PML inhibitor provided by the embodiments of the present application can inhibit the level of PML protein up-regulated by RSL3, and thus in some embodiments can be further used to inhibit RSL3-induced cell senescence through CP.

[0064] In some embodiments, the PML inhibitor can be used to inhibit the formation of PML nuclear bodies (Promyelocytic leukemia-nuclear bodies, PML NBs). RSL3 can promote the formation of PML NBs, and CP can inhibit the formation of PML NBs caused by RSL3. That is, the PML inhibitor can reduce the formation of PML nuclear bodies induced by RSL3 through CP, thereby producing an inhibitory effect.

[0065] Related studies have reported that PML is involved in the regulation of cell senescence (see the following literature: Ivanschitz L, De Thé H, Le Bras M. PML, SUMOylation, and Senescence. Front Oncol. 2013 Jul 4; 3: 171. doi:

[0066] 10.3389 / fonc.2013.00171.) and PML regulates p53 cell senescence pathway by modulating post-translational modification processes through the formation of PML nuclear bodies (Promyelocytic leukemia-nuclear bodies, PML NBs) (see the following reference: Lallemand-Breitenbach V, de Thé H. PML nuclear bodies: from architecture to function. Curr Opin Cell Biol. 2018 Jun;52:154-161. doi: 10.1016 / j.ceb.2018.03.011.). Therefore, the number of PML NBs can reflect the expression level of PML. Since CP can inhibit the formation of PML NBs induced by RSL3, it can be understood that CP can inhibit the expression of PML protein.

[0067] In some embodiments, the concentration of CP in the PML inhibitor is 2-500 μM, optionally 50-200 μM, optionally 100 μM or 200 μM.

[0068] <CP for use in inhibiting cell senescence>

[0069] The embodiments of the present application also provide a use of CP in the preparation of a cell senescence inhibitor.

[0070] The cell senescence inhibitor can inhibit the level of PML protein up-regulated by RSL3 induction by CP, and can be used to inhibit RSL3-induced cell senescence.

[0071] In some embodiments, the concentration of CP in the cell senescence inhibitor is 2-500 μM, optionally 50-200 μM, optionally 100 μM or 200 μM.

[0072] <GPX4 binding preparation>

[0073] The embodiments of the present application provide a GPX4 binding preparation comprising CP.

[0074] In some embodiments, the GPX4 binding preparation comprises DMSO.

[0075] In some embodiments, the GPX4 binding preparation is used for the enzymatic activity of GPX4.

[0076] In some embodiments, the GPX4 binding preparation is used for inhibiting the expression of PML protein.

[0077] In some embodiments, the GPX4 binding preparation is used for inhibiting the formation of PML nuclear bodies.

[0078] In some embodiments, the GPX4-binding agent is used to inhibit cell senescence.

[0079] In some embodiments, the concentration of CP in the GPX4-binding agent is 2-500 µM, optionally 50-200 µM, optionally 50, 100 or 200 µM.

[0080] <inhibitor>

[0081] An inhibitor is provided in embodiments of the present application, comprising CP.

[0082] The inhibitor is a PML inhibitor.

[0083] In some embodiments, the PML inhibitor is used to inhibit PML protein expression.

[0084] In some embodiments, the PML inhibitor is used to inhibit PML nuclear body formation.

[0085] In some embodiments, the PML inhibitor is used to inhibit cell senescence.

[0086] In some embodiments, the concentration of CP is 2-500 µM, optionally 50-200 µM, optionally 100 µM or 200 µM.

[0087] <Experimental Example 1, LC-MS determination of GPX4 enzyme activity in cells>

[0088] 1.1 Experimental method

[0089] The method of Experimental Example 1 is shown in the following steps S101-S104:

[0090] Step S101, obtain glutathione peroxidase 4 (GPX4) overexpressed HEK293 cell solution.

[0091] Step S101 is specifically as follows:

[0092] Plate HEK293 cells with a density of 70-80% for transfection;

[0093] Add 1.5 mL of opti-MEM medium, 25 μg of GPX4 plasmid (the GPX4 plasmid is a plasmid carrying the GPX4 gene, and the GPX4 plasmid is constructed by Kai Life Company) and 25 μL of neofect TM (neofect TM is a transfection reagent, which is derived from a transfection kit, and the transfection kit is specifically Neofect TMDNA transfection reagent, purchased from Lingkechuangzhi (Beijing) Biotechnology Co., Ltd., was mixed uniformly for transfection, incubated at room temperature for 15 minutes, then 25 mL of complete culture medium without secondary antibody was added to a super large dish to obtain the incubated mixture, and the incubated mixture and the aforementioned HEK293 cells with a density of 70% to 80% were added to the dish; then the dish was incubated in an incubator for 24 hours to collect the cells, thereby obtaining GPX4 overexpression HEK293 cell liquid, and Western blotting (WB) was used to detect the GPX4 overexpression HEK293 cell liquid, and the results are shown in Figure 2 .

[0094] Step S102, obtaining crude enzyme liquid.

[0095] Step S102 is specifically as follows:

[0096] The culture medium in the GPX4 overexpression HEK293 cell liquid was removed, 1 mL of PBS buffer was added for washing, the old PBS buffer was removed, 1 mL of new PBS buffer was added, the cells were collected with a cell scraper, the collected cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, 100-200 μL of PBS buffer was added, the cells were suspended, and the cells were repeatedly frozen and thawed at a temperature of-80℃ and room temperature for 3 times, ultrasonic crushing was performed for 30 s (the power of ultrasonic was 5 Hz), then centrifugation was performed at a speed of 12000 rpm for 15 min, the supernatant was taken, the protein content was determined, and the crude enzyme liquid was prepared for standby.

[0097] Step S103, setting control group, experimental groups 1-3, and preparing corresponding reaction buffer for each group.

[0098] The reaction buffer of step S103 is specifically as follows:

[0099] The total volume of the reaction buffer is 100 μL, which includes 100 mM Tris-HCl (pH = 7.4), 2 μM PE-OOH (phosphatidylethanolamine hydroperoxide), 0.1% Triton X-100, 5 mM EDTA (ethylenediaminetetraacetic acid), 3 mM GSH (glutathione), 1.5 mM NADPH (reduced coenzyme II, scientific name: reduced nicotinamide adenine dinucleotide phosphate) and crude enzyme liquid.

[0100] Step S104, reaction processing according to the reaction buffer.

[0101] To the reaction buffer of the experimental groups 1-3, CP was added respectively, so that the concentration of CP was 125 μM, 250 μM and 500 μM respectively, and no CP was added to the reaction buffer of the control group;

[0102] Then, each group was reacted at a temperature of 37℃ for 30 min, and after the reaction was completed, 600 μL of chloroform-methanol (the volume ratio of chloroform to methanol was 2:1) was added to terminate the reaction, the lower layer was taken out, nitrogen was blown dry, 50 μL of methanol was added for redissolution, 2 μL of an internal standard (the internal standard was PEOOH) was added, and centrifugation was performed at a temperature of 4℃ and a speed of 14000 rpm for 30 min, and the supernatant was taken as the test liquid of each group.

[0103] Step S104, detecting the GPX4 enzyme activity of the test liquid of each group by liquid chromatograph-mass spectrometer (LC-MS).

[0104] The liquid phase analysis conditions of LC-MS in step S104 are as follows:

[0105] The chromatographic column was Waters Premier A CQUITY BEH HILIC (specification: 100×2.1 mm, 1.7 μm), the column temperature was 40℃, the mobile phase included phase A and phase B, phase A was a mixture of acetonitrile, water and ammonium formate, the volume ratio of acetonitrile to water was 95:5, and the concentration of ammonium formate was 10 mM, and phase B was a mixture of acetonitrile, water and ammonium formate, the volume ratio of acetonitrile to water was 50:50, and the concentration of ammonium formate was 10 mM.

[0106] Gradient elution: 0-10.0 minute, 0-35% B phase; 10.0-10.1 minute, 35-100% B phase; 10.1-13.0 minute, 100% B phase; 13.0-13.1 minute, 100-0% B phase; 13.1-22.0 minute, 0% B phase;

[0107] The flow rate was 0.25 mL / min;

[0108] The injection amount was 2 μL.

[0109] The mass spectrometry analysis conditions of LC-MS in step S104 are as follows:

[0110] The ion source was an electrospray ionization source (ESI);

[0111] The spray voltage was negative ion, -2.8 KV;

[0112] The lens voltage was 65;

[0113] The transmission capillary temperature was 350℃;

[0114] Heating temperature of auxiliary gas: 320℃;

[0115] V-Sheath gas: 30 arb;

[0116] W-Auxiliary gas: 15 arb;

[0117] X-Backflush gas: 0 arb;

[0118] Activity calculation: Calculate the generation rate of PE-OH (phosphatidylethanolamine hydroxylate) within 30 min, which represents the enzyme activity of GPX4.

[0119] 1.2 Experimental results and analysis

[0120] The experimental results are shown in Figures 2-3 .

[0121] Figure 2 The GPX4 overexpression effect of GPX4 overexpression HEK293 cell liquid is shown.

[0122] Figure 2 GAPDH in refers to glyceraldehyde-3-phosphate dehydrogenase, which is the most commonly used loading internal reference in WB experiments; Vector refers to a blank vector, which plays a role similar to a blank control; OE-GPX4 refers to GPX4 after transfection. From the WB detection results of Figure 2 , it can be seen that the expression of GPX4 after transfection is excessive, which proves that the overexpression is successful.

[0123] Figure 3 The GPX4 enzyme activity (GPX4 activity) schematic diagram of the control group and experimental groups 1-3 is shown.

[0124] In order to detect the effect of CP on the enzyme activity of GPX4, experimental example 1 uses LC-MS technology to detect the activity of GPX4 in cells. First, overexpress GPX4 on HEK293 cells, and verify the overexpression effect of GPX4 by Western blotting Figure 2 ). Then collect the crude enzyme of GPX4 in the cells, add PEOOH as the reaction substrate, under certain reaction conditions, by detecting the generation rate of product PE-OH, reflecting the enzyme activity of GPX4.

[0125] As shown in Figure 3 , when the concentration of CP is 125 μM and 250 μM, there is no obvious activation effect, and when the concentration is 500 μM, it has obvious activation effect on the enzyme activity of GPX4.

[0126] <Experimental example 2, cell heat migration analysis>

[0127] 2.1 Experimental method

[0128] Experimental Example 2 uses a cellular thermal shift assay (CETSA) to explore whether CP can bind to GPX4. The principle of the CETSA experiment is that as the temperature rises, the protein denatures, the solubility decreases, and the protein precipitates out of the solution, resulting in a decrease in the content of soluble protein in the solution; when a small molecule binds to the protein, it will have a certain stabilizing effect on the spatial structure of the protein, so that the content of non-denatured protein increases under the same temperature conditions, thereby causing the solubility curve of the protein to shift to the right.

[0129] The method of Experimental Example 2 is as shown in the following steps S201-S207:

[0130] Step S201, expand HEK293 cells in the culture wells of a six-well plate (having 6 culture wells) to a cell confluence of 90%;

[0131] Step S202, after washing the cells with a PBS buffer twice, add 1 mL of PBS buffer to each culture well, collect the cells with a cell scraper, and centrifuge the collected cells at a speed of 1000 rpm for 5 min to obtain a cell precipitate;

[0132] Step S203, add a mixed PBS buffer containing protease inhibitors (400 uL of the mixed PBS buffer is mixed by 396 uL of a PBS buffer and 4 uL of a protease inhibitor) to the cell precipitate, repeat freezing and thawing at temperatures of 25°C and -80°C for 3 times, each time for 30 min, then centrifuge at a speed of 12000 rpm for 15 min to obtain a supernatant, and determine the protein content by the BCA method;

[0133] Step S204, divide the supernatant into a control group (i.e., the Control group) and a drug administration group (i.e., the CP group); for the drug administration group, add DMSO and CP to the supernatant to obtain samples of the drug administration group, and the concentration of CP in the samples of the drug administration group is 50 mM; for the control group, add DMSO (the amount of DMSO added to the control group is equal to that of the CP group) to the cell lysate to obtain samples of the control group; then incubate the samples of the control group and the drug administration group at 37°C for 1 h, and then divide each group of samples into 10 equal parts and load them into corresponding sample tubes (each sample tube contains 1 part of the corresponding sample);

[0134] Step S205, after the completion of step S204, set temperature gradients of 45°C, 49°C, 53°C, 57°C, 61°C, 65°C, 69°C, 73°C, 77°C, and 81°C according to the 10 samples of each group, and place the samples in each sample tube of each group in the corresponding different temperatures, and set the incubation time of each temperature gradient to 4 min for temperature rising treatment;

[0135] Step S206, after the temperature rising is completed, the samples in each group of sample tubes are placed at room temperature for 3 min, and centrifuged at a speed of 14000xg for 20 min;

[0136] Step S207, after centrifugation, the supernatant is taken, and the content of GPX4 and GAPDH protein (used as an internal reference protein) in each group at different temperatures is detected by Western blotting experiment method, and the corresponding protein thermal stability curve is drawn according to the content.

[0137] 2.2 Experimental results and analysis

[0138] The experimental results are shown in Figures 4-6 .

[0139] Figure 4 An illustration of the content of GPX4 and GAPDH protein at different temperatures in the administration group and the control group is shown;

[0140] Figure 5 An illustration of the protein thermal stability curve of GPX4 protein at different temperatures in the administration group and the control group is shown; the vertical coordinate represents the relative signal intensity (Relative intensity), and the horizontal coordinate represents the temperature (temperature, unit: ℃).

[0141] Figure 6 An illustration of the protein thermal stability curve of GAPDH protein at different temperatures in the administration group and the control group is shown; the vertical coordinate represents the relative signal intensity (Relative intensity), and the horizontal coordinate represents the temperature (temperature, unit: ℃).

[0142] It should be noted that the relative signal intensity (Relative intensity) reflects the content of the protein.

[0143] As can be seen from Figure 5 , compared with the control group, the dissolution curve of GPX4 in the administration group is right-shifted due to CP treatment, indicating that CP improves the thermal stability of GPX4. Therefore, CP can have a direct interaction with GPX4 in the cell. Under the premise of having a combination, CP can play a role in improving the enzyme activity of GPX4.

[0144] <Experiment Example 3, Microscale Thermophoresis (MST) Analysis>

[0145] 3.1 Experimental method

[0146] Experimental Example 3 uses MicroScale Thermophoresis (MST) experiment to explore whether CP can form a complex with GPX4. The principle of the MST experiment is: using infrared laser to irradiate the sample in the capillary, so that the water molecules in the sample absorb infrared light and heat, forming a temperature gradient. Biological molecules undergo directional movement in this temperature gradient, and the changes in mass, charge, hydration layer and conformation of biological molecules will affect their movement speed in the temperature gradient field, which can be used to analyze the interaction between molecules.

[0147] The experimental method of Experimental Example 3 is shown in the following steps S301-S304:

[0148] S301, obtaining labeled glutathione peroxidase 4 (GPX4).

[0149] The GPX4 protein labeling is performed according to the procedure of the Monolith protein labeling kit RED-NHS Generation 2 kit.

[0150] S302, mixing CP and DMSO to form CP solutions of different concentrations (the concentrations of CP are 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625, 0.48828, 0.24414 μM, respectively); mixing different concentrations of CP solution with labeled GPX4, and incubating at room temperature for 30 min to obtain incubated samples containing different concentrations of CP.

[0151] S303, irradiating the incubated sample as the sample to be measured;

[0152] Specifically, the sample is irradiated using a light-emitting diode, and the irradiation conditions are: the intensity of the light-emitting diode is set to 20%, the excitation power is set to 5%, and the temperature is set to 25°C.

[0153] S304, loading and measuring the sample on a biological molecule interaction analyzer instrument (specifically, NanoTemper Monolith instrument NT.115), and analyzing the data using MO.Affinity Analysis (x86) software, and recording the results.

[0154] 3.2 Experimental results and analysis

[0155] After detection, the signal-to-noise ratio (Singal to Noise, S / N) is measured to be 5.2, which is greater than 5 (i.e., S / N>5), indicating that CP can form a complex with GPX4. Under the premise of complex formation, CP can possibly play a role in improving the enzyme activity of GPX4.

[0156] In addition, the results of measuring the fluorescence value of the sample with the change of the concentration of CP are shown in the following table 2. Figure 7 Figure 7 An example of analyzing the binding ability of CP and GPX4 by MST is shown in the following table 3.

[0157] Figure 7 In the table 3, Fnorm refers to the fluorescence value; Ligand concentration refers to the concentration of the ligand, that is, the concentration of CP.

[0158] Figure 7 In the table 3, Kd is the equilibrium dissociation constant, and the KD value represents the concentration of the Ligand molecule (i.e., the CP molecule) when 50% of the Target molecule (i.e., the GPX4 molecule) is combined, and the unit is M (mol / L, mol per liter). The larger the KD value, the lower the affinity, and vice versa. The smaller the KD value, the higher the affinity between the Target molecule and the Ligand molecule, and the stronger the binding ability.

[0159] From the table 3, it can be seen that the initial fluorescence value changes regularly with the ligand concentration, and the KD value is 25.69 μM, indicating that the affinity of CP and GPX4 is high, and the binding ability is good. Figure 7

[0160] <Experiment Example 4, Detection of PML Protein Expression in HDFs Cells>

[0161] 4.1 Experimental method

[0162] Experiment Example 4 detects the expression of PML protein in HDFs cells, and the specific method is shown in the following steps S401-S404:

[0163] Step S401, set up a control group, a model group, an experimental group 1 and an experimental group 2, and configure corresponding cell culture holes for each group on a 6-hole plate, evenly spread HDFs cells into the 6-hole plate (add 2 mL of culture medium per hole), and configure corresponding cells to be treated for each group, then shake gently, and put into a cell culture box for 24 h.

[0164] Step S402, when the cell confluence is about 50%, start dosing for the model group, experimental group 1-2, and the cell amount is about 10 5 cells, the model group, experimental group 1-2, add corresponding dosing according to the following table 1, remove after 6 h, replace with fresh culture medium for 4 days, and replace the liquid every 2 days during the culture period; the control group does not add dosing.

[0165] Table 1

[0166] ​​

[0167] Note: RSL3 is an inhibitor of glutathione peroxidase 4 (GPX4)

[0168] Step S403, after the treatment of step S402, the cells of each group were collected and 400 μL of appropriate cell lysis solution (product number P0013J, brand Biyun Tian, purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) was added. Lysis in ice bath for at least 30 min, centrifuged at 12000xg, 4℃ for 10 min, take the supernatant, according to the BCA kit to determine the protein concentration.

[0169] Step S404, take the supernatant obtained in step S403, detect the expression level of PML protein.

[0170] Step S404 is as follows:

[0171] Take the supernatant obtained in step S403, and perform protein denaturation treatment in a metal bath for 10 minutes. After denaturation of the protein, perform SDS-PAGE electrophoresis, and wet transfer for 60 min.

[0172] Then incubate with PML primary antibody and GAPDH primary antibody at 4℃ overnight.

[0173] Then incubate with secondary antibody at room temperature for 2h.

[0174] Then use a chemiluminescence workstation to perform imaging, and record the imaging results, as shown in Figure 8 Image J software is used to analyze the image, and the results are expressed as the optical density ratio of the target protein (PML protein) to the internal reference protein GAPDH. The optical density ratio is visualized by intensity, as shown in Figure 9

[0175]

[0176] 1 μL of PML primary antibody stock solution (from Wuhan Sanying Biotechnology Co., Ltd. (referred to as proteintech), product number: 21041-1-AP) is added to 1 mL of TBST to obtain the aforementioned PML primary antibody.

[0177] 1 μL of GAPDH primary antibody stock solution (from Hangzhou Fude Biotechnology Co., Ltd., product number: FD0063) is added to 3 mL of TBST to obtain the aforementioned GAPDH primary antibody.

[0178] 1 μL of secondary antibody stock solution (from Hangzhou Fude Biotechnology Co., Ltd., product number: FDR007) is added to 3 mL of TBST to obtain the aforementioned secondary antibody.

[0179] 4.2 Experimental results and analysis

[0180] ​​From the above, experimental example 4 detected the protein expression level of PML in each group of cells by western blot (immunoblotting), and the results are shown in Figures 8-9 .

[0181] Figure 8 a relative expression level of PML protein and GAPDH protein in each group;

[0182] Figure 9 a relative gray value ratio of PML protein and GAPDH protein in each group.

[0183] According to Figures 8-9 , it can be seen that:

[0184] Compared with the control group (normal group), the PML protein in the model group was significantly up-regulated, indicating that PML was very sensitive to lipid peroxidation caused by GPX4 inhibition; combined with the results of experimental groups 1-2, it can be seen that CP can reverse the increase in the expression level of PML protein induced by RSL3.

[0185] In summary, CP can reduce the level of PML protein up-regulated by RSL3, and can be used to inhibit RSL3-induced cell senescence.

[0186] <Experimental Example 5, Immunofluorescence Staining to Detect the Expression of PML in Cells>

[0187] 5.1 Experimental method

[0188] Experimental example 5 used a laser confocal microscope to observe the expression of PML, and the specific method is shown in the following steps S501-S507:

[0189] Step S501, take the cell liquid containing HDFs cells (the cell liquid is mixed by the culture medium and HDFs cells), divide into control (control) group, experimental group 1 and experimental group 2, experimental group 1 is treated with RSL3 for 6h, experimental group 2 is treated with RSL3 and CP with a concentration of 100μM for 6h, and the control group is not treated.

[0190] Step S502, cell fixation: remove the culture medium of each group, wash with PBS for 3 times, use 1mL of fixing solution (the fixing solution is paraformaldehyde) to fix at room temperature for 15min, wash with PBS buffer for 3 times, and get the fixed cells of each group.

[0191] Step S503, blocking treatment: 1 mL of blocking solution (the blocking solution is a liquid diluted by PBS, which contains 5% BSA and 0.2% Triton x-100, BSA is bovine serum albumin, and Triton x-100 is Triton X100) is added to each group of fixed cells, and the cells are blocked at room temperature for 1 h, washed with PBS buffer for 3 times, and finally completely removed with PBS buffer to obtain blocked cells, which are stored in the dish.

[0192] Step S504, primary antibody incubation: 100 μL of primary antibody diluent (PML (rabbit), the primary antibody diluent is diluted by PBS at a ratio of 1:1000) is added to the bottom center of each group of dishes, and then the dishes are placed in a wet box and incubated at 4°C overnight to obtain the dishes after primary antibody incubation.

[0193] Step S505, secondary antibody incubation: 100 μL of secondary antibody diluent (Alexa Fluor 488-conjugated Goat anti-rabbit IgG is diluted by PBS at a ratio of 1:500) is added to the bottom center of the dishes after primary antibody incubation, and then the dishes are incubated at room temperature for 2 h, washed for 3 times, and finally completely removed with PBS buffer to obtain the cells after secondary antibody incubation.

[0194] Step S506, nucleus staining: 100 μL of DAPI diluent (the concentration of DAPI in the DAPI diluent is 5 μg / mL, and the DAPI diluent is diluted by PBS at a dilution ratio of 1:1000 from the DAPI stock solution, DAPI stock solution, item number C1002, brand Biyun Tian) is added to the bottom center of the dishes after secondary antibody incubation, and then the dishes are incubated at room temperature in the dark for 10 min, washed with PBS for 3 times, and then 1 mL of PBS buffer is added to obtain the stained cells of each group.

[0195] Step S507, each group of stained cells is observed by using a laser confocal microscope, and the results are recorded.

[0196] 5.2 Experimental results and analysis

[0197] The experimental results are shown in Figure 10 .

[0198] Figure 10 is a fluorescence schematic diagram of the stained cells of each group.

[0199] Figure 10In the table, the PML row pictures correspond to the images of immunofluorescence staining of PML protein (red); the Merge row pictures correspond to the "fusion" images; the results of immunofluorescence staining of PML protein (red) and the results of staining of cell nucleus (commonly stained by DAPI or other dyes into blue, the blue part in the figure is the cell nucleus) in the same scene are combined into one picture, that is, the "fusion" images corresponding to the Merge row pictures can be obtained. Figure 10 The position of PML protein in the cell and the spatial relationship between the position and the cell nucleus can be presented.

[0200] According to Figure 10 It can be seen that, compared with the control group, the number of PML NBs in the nucleus of the cells treated by RSL3 for 6h in the experimental group 1 significantly increased, indicating that RSL3 promoted the formation of PML NBs.

[0201] In the experimental group 2, the cells were co-incubated with CP (100 μM) and RSL3 for 6h, and compared with the experimental group 1 in which RSL3 was added alone, the number of PML NBs significantly decreased, indicating that CP could inhibit the formation of PML NBs.

[0202] Therefore, CP can inhibit the aggregation and formation of PML NBs, and can be used to protect the cell senescence induced by RSL3.

[0203] The above detailed description has described the present application in detail, but these do not constitute limitations on the present application. The protection scope of the present application is not limited to the above embodiments, and any equivalent modifications or changes made by those skilled in the art according to the disclosed content of the present application shall be included in the protection scope recited in the claims.

Claims

1. Use of cafestol palmitate for the manufacture of a GPX4 binding agent.

2. Use according to claim 1, the GPX4 binding agent for activating GPX4 activity.

3. Use according to claim 1, the GPX4 binding agent for inhibiting PML protein expression.

4. Use according to claim 1, the GPX4 binding agent for inhibiting PML nucleosome formation.

5. Use of cafestol palmitate for the manufacture of a GPX4 activity activating agent.

6. Use of cafestol palmitate for the manufacture of a PML inhibitor.

7. Use according to claim 6, the PML inhibitor for inhibiting PML protein expression, and / or inhibiting PML nucleosome formation, and / or inhibiting cellular senescence.

8. Use of cafestol palmitate for the manufacture of a cellular senescence inhibitor.

9. A GPX4-binding formulation characterized in that, comprising cafestol palmitate.

10. A PML inhibitor characterized in that, comprising cafestol palmitate.

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