Autophagosome, extraction method therefor and use thereof

Abstract

Provided are an autophagosome, an extraction method therefor and the use thereof. Provided is a method for extracting a new extracellular vesicle that inhibits ferroptosis, i.e., an autophagosome, which has the advantages of being easy to acquire and not requiring special stimulation for maternal cells, is suitable for large-scale extraction, and has a powerful clinical potential application value. Also provided is an autophagosome obtained using the above extraction method. In examples, a fibroblast ferroptosis model is constructed using high concentration glucose, and the autophagosome is used for verification. It is confirmed that the autophagosome can reduce the sensitivity of cells to ferroptosis and enable the cells to escape from adverse factors inducing ferroptosis.

Description

Autophagosome, extraction method and application thereof Technical Field

[0001] The present application belongs to the field of biopharmaceutical technology, and specifically relates to an autophagosome and an extraction method and application thereof. Background Art

[0002] Ferroptosis is a newly discovered cell-mediated ferrous ion (Fe 2+ ) overload and iron-dependent lipid peroxide accumulation. Glutathione peroxidase 4 (GPX4) is an antioxidant enzyme negatively regulated by endoplasmic reticulum (ER) stress that can clear excess lipid peroxides and is a key upstream regulator of ferroptosis. However, under high glucose conditions, the content and activity of GPX4 protein in cells are significantly downregulated, leading to reactive oxygen species (ROS) and Fe 2+ Ferritin heavy chain 1 (FTH1) is an iron storage protein that reversibly stores free Fe 2+ , regulating intracellular free Fe 2+ concentration, reducing the Fe 2+ However, the expression level of FTH1 protein in renal tubular cells (HK-2 cells) was significantly downregulated under high glucose conditions.

[0003] Autophagosomes, also known as secretory autophagosomes (SAPs), are produced by secretory autophagy and belong to the family of extracellular vesicles (EVs). They are a new mode of intercellular communication by transferring cargo to target cells. Exposure to stress and starvation can enhance secretory autophagy and reduce degradative autophagy by inducing the loss of lysosomal integrity. In recent years, research on SAPs has mainly focused on clarifying their role in the occurrence and development of diseases such as cancer and acute respiratory distress syndrome (ARDS), providing a promising strategy for the diagnosis and mechanism research of these diseases. Although the pathological effects of SAPs on certain diseases have been described, the therapeutic application of SAPs has not yet been explored, especially in the field of ferroptosis.

[0004] Summary of the Invention

[0005] The purpose of the present application is to provide an autophagosome and its extraction method and application, wherein the autophagosome can significantly protect cells from ferroptosis or reduce sensitivity to ferroptosis, and can be used as a ferroptosis inhibitor.

[0006] The present application provides a method for extracting autophagosomes, comprising the following steps: culturing vascular endothelial cells in a starvation environment for a period of time, subjecting the cell supernatant to low-speed centrifugation to obtain a supernatant, and then subjecting the supernatant to high-speed centrifugation to obtain a precipitate; the centrifugal force of the low-speed centrifugation is 2000g, and the centrifugal force of the high-speed centrifugation is 12000g;

[0007] The precipitate is incubated with LC3-labeled magnetic beads to capture autophagosomes.

[0008] Preferably, the starvation environment comprises DMEM medium without exosome fetal bovine serum.

[0009] Preferably, the vascular endothelial cells are cultured in DMEM medium without exosome fetal bovine serum for 48 to 72 hours.

[0010] Preferably, the low-speed centrifugation time is 10 to 15 minutes; the high-speed centrifugation time is 15 to 20 minutes.

[0011] Preferably, the incubation temperature is room temperature, and the incubation time is 30 minutes to 2 hours.

[0012] Preferably, after the incubation, the method further comprises eluting the autophagosomes using an elution solution.

[0013] The present application also provides autophagosomes obtained using the above extraction method.

[0014] The present application also provides the use of the above-mentioned autophagosome in the preparation of a cell ferroptosis inhibitor.

[0015] Preferably, the cell ferroptosis includes cell ferroptosis induced under high glucose conditions.

[0016] The present application also provides a cell ferroptosis inhibitor, comprising the above-mentioned autophagosome and auxiliary materials.

[0017] Beneficial effects: This application provides a new type of extracellular vesicle that inhibits ferroptosis, namely autophagosomes, and a method for extracting the same. It has the advantages of being easy to obtain and not requiring special stimulation of the parent cells, is suitable for large-scale extraction, has strong potential clinical application value, and provides a new strategy for inhibiting cell ferroptosis in the future. In the examples of this application, a fibroblast ferroptosis model was constructed using high sugar, and the autophagosomes were used for verification, confirming that the autophagosomes can reduce the sensitivity of cells to ferroptosis, allowing cells to escape adverse factors that induce ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 is a diagram showing the expression of iron ions and ROS in high glucose-induced fibroblasts (HG-HDFs) and their effects on the expression of ferroptosis-related genes GPX4 and FTH1, provided in Example 1 of the present application;

[0019] FIG2 is a graph showing the expression results of surface-specific marker proteins of autophagosomes provided in Example 2 of the present application;

[0020] FIG3 is a particle size analysis diagram of the autophagosome provided in Example 2 of the present application;

[0021] FIG4 is an electron micrograph of an autophagosome provided in Example 2 of the present application;

[0022] FIG5 is an evaluation and analysis diagram of the effect of autophagosomes on iron metabolism, peroxidation, and expression of ferroptosis-related proteins in ferroptosis model cells provided in Example 3 of the present application;

[0023] FIG6 is a graph showing the effect of SAPs on cell viability of fibroblasts;

[0024] FIG7 shows the effects of SAPs on endoplasmic reticulum stress-related proteins and proteins that inhibit ferroptosis after TM pretreatment;

[0025] Figure 8 shows the effect of SAPs on the exosome content in the supernatant of HG-HDFs cells. DETAILED DESCRIPTION

[0026] The present application provides a method for extracting autophagosomes, comprising the following steps: culturing vascular endothelial cells in a starvation environment for a period of time, subjecting the cell supernatant to low-speed centrifugation to obtain a supernatant, and then subjecting the supernatant to high-speed centrifugation to obtain a precipitate; the centrifugal force of the low-speed centrifugation is 2000g, and the centrifugal force of the high-speed centrifugation is 12000g;

[0027] The precipitate is incubated with LC3-labeled magnetic beads to capture autophagosomes.

[0028] The present application cultured vascular endothelial cells for a period of time in a starvation environment, preferably comprising DMEM medium without exosomes and fetal bovine serum. In the examples of the present application, the vascular endothelial cells were preferably cultured in DMEM medium without exosomes and fetal bovine serum for 48 hours, and the cell supernatant was collected. The present application stimulated the production of secretory autophagosomes in vascular endothelial cells under a starvation environment and amplified the starvation autophagosomes in large quantities.

[0029] After obtaining the supernatant, the present application performs low-speed centrifugation on the supernatant, the centrifugal force of the low-speed centrifugation is 2000g, and the time of the low-speed centrifugation is preferably 10 to 15 minutes, and the supernatant is recovered and the precipitate (cell debris and impurities) is removed, and then the supernatant is extracted.

[0030] The present invention is to perform high-speed centrifugation on the supernatant after low-speed centrifugation, preferably at 12000g for 15-20 minutes, to obtain a precipitate. In a specific embodiment of the present invention, to ensure purity, the precipitate can also be resuspended in sterile PBS buffer and then centrifuged again at 12000g for 15-20 minutes to obtain a precipitate.

[0031] The present application preferably uses magnetic beads with surface-labeled LC3 antibodies to incubate and adsorb cell pellets resuspended in PBS. The incubation temperature is preferably 37°C, and the time is preferably 30 minutes to 2 hours. The present application also preferably includes elution with an eluent after the incubation, and the eluent is preferably 0.1M NaOH.

[0032] The present application also provides autophagosomes obtained using the above extraction method.

[0033] The mean and peak values ​​of the particle size of the autophagosomes described in the present application are concentrated around 420 nm. They appear as a double-layer membrane-like structure under a transmission electron microscope, with an overall spherical or cup-shaped appearance and express LC3.

[0034] The present application also provides the use of the above-mentioned autophagosome in the preparation of a cell ferroptosis inhibitor.

[0035] The cell ferroptosis described in this application preferably includes cell ferroptosis induced under high sugar conditions. The embodiment establishes an HG-HDFs ferroptosis model. After different concentrations of autophagosomes are applied to HG-HDFs for 24 hours, the expression of intracellular free iron ions, reactive oxygen species, and ferroptosis-related proteins are detected. The results show that the addition of SAPs can significantly downregulate the level of free iron ions and reactive oxygen species in fibroblasts, the disappearance of mitochondrial cristae induced by ferroptosis can also be restored by autophagosomes, and the expression of FTH1 protein and GPX4 protein that can inhibit ferroptosis is significantly decreased. The above results suggest that SAPs has the effect of inhibiting cell ferroptosis, and the effective concentration is 3mM, and the optimal concentration is 6mM.

[0036] The present application also provides a cell ferroptosis inhibitor, comprising the above-mentioned autophagosome and auxiliary materials.

[0037] In order to further illustrate the present application, an autophagosome provided by the present application and its extraction method and application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0038] Example 1

[0039] Construction of HG-HDFs ferroptosis model

[0040] Fibroblasts were cultured in glucose environments of LG (5mM), MG (5mM) and HG (50mM), respectively, and passaged normally. After 60 days, the expression of free divalent iron, peroxidation and ferroptosis-related proteins in fibroblasts in different glucose environments were detected.

[0041] 1. Method for detecting free divalent iron ions and peroxide:

[0042] (1) Determination of Fe 2+ HDFs treated with different concentrations of glucose were grown in 24-well plates. After the cells attached to the wall, they were incubated with 10 μM iron orange (red fluorescence) at 37°C for 30 min.

[0043] (2) The detection method of ROS is basically the same as that of Fe 2+ The reagent was replaced with 10 μM DHE and incubated at 37°C for 30 min. The cell nuclei were counterstained with Hoechst 33342. Typical images were taken by fluorescence microscopy.

[0044] 2. Detect the expression of ferroptosis-related proteins as follows:

[0045] (1) Protein concentration detection by BCA method: about 50 μL of the autophagosome suspension prepared by the method described in Example 2 and the cells derived from the autophagosomes were added with 50 μL of RIPA lysis buffer (R0010, Solebo Company), fully lysed at 4°C for 15 minutes, centrifuged (12000g, 4°C) for 20 minutes (cell lysate was prepared from 1 6-well plate of cells using 100 μL of RIPA lysis buffer), and the supernatant was slowly aspirated with a pipette and placed in a 1.5 mL EP tube; the protein concentration of the cell lysate and autophagosomes was determined using the BCA protein concentration detection kit (PC0020, Solebo Company), and the two groups of samples were diluted to the same concentration with PBS according to calculation, and the two groups were fully mixed according to the volume ratio of protein sample to loading buffer (5×) of 4:1, and boiled in an iron bath at 105°C for 10 minutes.

[0046] (2) Electrophoresis: Operate according to the instructions of the gel preparation kit. The lower gel is the separation gel and the upper gel is the concentration gel. After adding the lower gel (separation gel), use anhydrous ethanol to flatten it. After adding the upper gel (concentration gel), insert the comb as soon as possible to avoid bubbles in the tank (the presence of bubbles affects the electrophoresis of the sample). After taking the sample out of the refrigerator, thaw it, vortex and spin it (because a small amount of water vapor will condense on the tube cap, affecting the sample concentration). The sample volume is selected according to the protein concentration, and the sample volume is 20μg / well. Rainbow markers are added to the sample slots on both sides of the sample as a reference for the position of the target protein. The order of loading is cell protein lysate and autophagosomes. Electrophoresis is carried out at room temperature, with a constant voltage of 70mV for concentration. After entering the separation gel, the constant voltage is 130mV, and electrophoresis is carried out to the position required by the experiment.

[0047] (3) Electrotransfer: Prepare the electrotransfer equipment (electrotransfer tank, ice, etc.) and reagents (electrotransfer solution configuration: 100 mL of 10× electrotransfer solution, 700 mL of pure water, and 200 mL of methanol per 1000 mL of electrotransfer solution). During transfer, activate the PVDF membrane by soaking it in methanol for more than 10 seconds. No bubbles should appear between the gel and the membrane. The electrotransfer solution can be pre-cooled in ice. In the ice tank, maintain a constant current of 300 mA for 1 hour (the time can be adjusted according to the size of the target protein).

[0048] (4) Blocking: Remove the PVDF membrane from the electroporation tank and soak it in skim milk (5%, 100 mL plus 5 g of milk powder) for more than 1 hour. After blocking, collect the milk and store it in a 4°C refrigerator. Use it to dilute the secondary antibody the next day.

[0049] (5) Primary antibody incubation: dilute LC3, GPX4, FTH1, and GAPDH antibodies with primary antibody diluent (1:1000, i.e., 1 mL of primary antibody diluent plus 1 μL of primary antigen solution), incubate the diluted antibodies in the blocked PVDF membrane, and shake slowly at 4°C overnight.

[0050] (6) Secondary antibody incubation: Recover the primary antibody, rinse the membrane with PBST (PBS + 0.1% Tween) or TBST (TBS + 0.1% Tween), and shake rapidly on a shaker three times for 10 minutes each time. Then, soak the membrane in the secondary antibody (diluted with 5% skim milk, ratio 1:10,000) and incubate at room temperature for 1 hour;

[0051] (7) Exposure: Prepare the luminescent solution, place the strips in an exposure machine (GE), drip the luminescent solution on them for exposure, and collect images. If necessary, use Image J software to analyze the grayscale value of the protein strips.

[0052] The results are shown in Figure 1. After high glucose treatment, a large amount of iron ions and ROS accumulated in the macrophages, and the expression of iron storage-related proteins FTH1 and GPX4 was significantly reduced. The ferroptosis cell model was successfully constructed.

[0053] Example 2

[0054] The specific steps for extracting autophagosomes are as follows:

[0055] (1) The supernatant of the starved endothelial cells was collected and centrifuged at a low speed of 2000 g for 10 min.

[0056] (2) The supernatant was then collected and centrifuged at 12,000 g for 15 min. The precipitate was resuspended in sterile PBS buffer and centrifuged again at 12,000 g for 15 min. The precipitate was then collected.

[0057] (3) The precipitate resuspended in PBS was then adsorbed with magnetic beads carrying LC3 antibodies to obtain purified autophagosomes, which were then eluted using elution buffer (0.1 M NaOH).

[0058] Example 3

[0059] The autophagosome preparation obtained in Example 2 was identified:

[0060] (1) After extracting the total protein of starved vascular endothelial cells and their autophagosomes, the protein concentration was detected using a BCA kit. Western blot experiments were performed on a 15% gel. After loading the sample, protein electrophoresis, transfer, blocking, and primary and secondary antibody incubation were performed. Finally, chemiluminescence imaging analysis was used to expose and photograph the expression levels of the surface-specific marker protein LC3 of exosomes in vascular endothelial cells and their autophagosomes. The results are shown in Figure 2. Compared with the Cell lysis group, the expression of LC3II in the SAPs group was significantly increased, while the expression of LC3I was decreased, indicating that the sample SAPs contained a higher level of autophagosome expression, indicating that the extraction of autophagosomes was successful.

[0061] (2) The concentration and particle size of autophagosomes were detected by the nanoparticle analysis system. The results are shown in Figure 3. The mean and peak values ​​of the particle size of autophagosomes are concentrated around 420 nm, which is consistent with the particle size distribution characteristics of autophagosomes.

[0062] (3) The morphology of autophagosomes was observed by transmission electron microscopy, and photographs and records were taken. The results are shown in Figure 4. Autophagosomes contain classic double-layer membrane-like structures, and the overall appearance is spherical or cup-shaped.

[0063] Example 4

[0064] The qualified autophagosomes were identified by the identification method described in Example 3, and a control test related to ferroptosis in HG-HDFs was conducted. The specific steps were as follows:

[0065] After different concentrations of autophagosomes were applied to HG-HDFs for 24 hours, the expression of intracellular free iron ions, reactive oxygen species, and ferroptosis-related proteins were detected. The specific methods are as described in Example 1.

[0066] The results are shown in Figure 5. The addition of SAPs can significantly downregulate the level of free iron ions and reactive oxygen species in fibroblasts. The disappearance of mitochondrial cristae induced by ferroptosis can also be restored by autophagosomes. The expression of FTH1 protein and GPX4 protein that can inhibit ferroptosis is significantly decreased. The above results suggest that SAPs has the effect of inhibiting cell ferroptosis.

[0067] Example 5

[0068] In order to verify the biocompatibility of SAPs, the present application conducted the following related experiments, and the specific operations are as follows:

[0069] HG-HDFs were plated in 96-well plates and then treated as follows: the control group was added with an equal amount of PBS, and the experimental groups were added with different concentrations of SAPs. After 24 hours, the cell viability was detected.

[0070] The results are shown in Figure 6. The introduction of SAPs gradually increased the cell viability of fibroblasts, but when the SAPs concentration was 12 mM, its cell viability gradually decreased compared with the 6 mM group.

[0071] Example 6

[0072] To further explore the mechanism by which SAPs inhibit ferroptosis in HG-HDFs, we conducted the following experiments. The specific procedures are as follows:

[0073] First, HG-HDFs were pretreated with the ER stress activator tunicamycin (TM), and then 6 mM SAPs were added. Proteins were extracted and the expression of ER stress-related proteins (HSPA4, P-elf2α, elf2α, DDIT3) and proteins that inhibit ferroptosis (GPX4, FTH1) were detected.

[0074] The results, as shown in Figure 7, show that TM pretreatment with SAPs attenuated the inhibitory effects on ER stress-related proteins and the elevation of ferroptosis-inhibiting proteins (GPX4 and FTH1) compared to the SAPs alone group, demonstrating that SAPs plays a key role in suppressing ER stress in inhibiting high-glucose-induced ferroptosis in fibroblasts. Furthermore, observation of cytoplasmic iron deposition in cells from each group revealed that TM-pretreated cells maintained elevated levels of free iron in the cytoplasm after SAPs treatment. This further confirms the crucial role of ER stress in SAPs' inhibition of ferroptosis in HG-HDFs.

[0075] Example 7

[0076] The effects of SAPs on exosome secretion in HG-HDFs and the role of exosome secretion in SAPs inhibiting ferroptosis in HG-HDFs were examined. The specific procedures were as follows:

[0077] First, the cell supernatants of HG-HDFs after 24 hours of SAPs treatment were collected, and the cell supernatants after an equal amount of PBS treatment were used as controls. The two groups of exosomes were extracted by ultracentrifugation. The exosome concentrations were detected by NTA, and the free iron ion content contained in the exosomes was detected by ICP-MS. Then, to further verify the role of exosome secretion in SAPs inhibiting HG-HDFs ferroptosis, HG-HDFs were pretreated with the exosome secretion inhibitor GW4869, and then intracellular free iron ion staining was performed after SAPs were added for 24 hours.

[0078] The results are shown in Figure 8. The content of exosomes in the supernatant of HG-HDFs cells significantly increased after SAPs treatment, and the free iron ions contained in the exosomes also increased significantly. Inhibiting exosome secretion significantly weakened the inhibitory effect of SAPs on free iron ions in the cytoplasm of HG-HDFs.

[0079] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. A method for extracting autophagosomes, characterized in that: The following steps are involved: The vascular endothelial cells are cultured in a starvation environment for a period of time, the cell supernatant is subjected to low-speed centrifugation to obtain a supernatant, and the supernatant is subjected to high-speed centrifugation to obtain a precipitate; the centrifugal force of the low-speed centrifugation is 1500-2000g, and the centrifugal force of the high-speed centrifugation is 10000-12000g; The autophagosomes were captured by incubating the precipitate with LC3 labeled magnetic beads.

2. The extraction method according to claim 1, characterized in that The centrifugal force of the low-speed centrifugation is 2000g, and the centrifugal force of the high-speed centrifugation is 12000g.

3. The extraction method according to claim 1, characterized in that The starvation environment includes a culture environment provided by a DMEM culture medium without exosome fetal bovine serum.

4. The extraction method according to claim 3, characterized in that: The vascular endothelial cells are cultured in a DMEM medium containing no exosome fetal bovine serum for 48 to 72 hours.

5. The extraction method according to claim 1, characterized in that: The low-speed centrifugation time is 10 to 15 minutes; The time of high-speed centrifugation is 15 to 20 minutes.

6. The extraction method according to claim 1, characterized in that: After obtaining the precipitate, the method further includes resuspending the precipitate with a sterile PBS buffer and centrifuging again at 12000 g for 15 to 20 minutes to obtain the precipitate.

7. The extraction method according to claim 1, characterized in that: The incubation temperature is 37° C., and the incubation time is 30 min to 2 h.

8. The extraction method according to claim 1 or 7, characterized in that After the incubation, the autophagosomes are eluted using an elution solution.

9. The extraction method according to claim 8, characterized in that: The eluent is 0.1 M NaOH solution.

10. Autophagosome obtained by the extraction method according to any one of claims 1 to 9.

11. The autophagosome according to claim 10, characterized in that The autophagosomes express LC3.

12. Use of the autophagosome according to claim 10 or 11 in the preparation of a cell ferroptosis inhibitor.

13. The use according to claim 12, characterized in that: The cell ferroptosis includes cell ferroptosis induced under high glucose conditions.

14. The use according to claim 12, characterized in that: The effects of the cell ferroptosis inhibitor include at least one of the following: (1) Significantly down-regulate the level of free iron ions in fibroblasts; (2) Significantly down-regulate the level of reactive oxygen species in fibroblasts; (3) restore ferroptosis-induced mitochondrial cristae; (4) The expression of FTH1 protein and / or GPX4 protein, which inhibit ferroptosis, is decreased.

15. A cell ferroptosis inhibitor, characterized in that It comprises the autophagosome according to claim 10 or 11 and auxiliary materials.

16. The cell ferroptosis inhibitor according to claim 15, characterized in that The working concentration of the autophagosome is not less than 3 mM.

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