Application of preparation for regulating ULK1 phosphorylation level in preparation of product for regulating autophagic flow of immune cells

Abstract

The invention provides application of a preparation for regulating ULK1 phosphorylation level in preparation of a product for regulating autophagic flow of immune cells, and belongs to the technical field of biological medicines. In particular to application of a preparation for regulating the phosphorylation level of ULK1 in preparation of a product for inhibiting autophagic flow of immune cells, and the preparation is an STING agonist. The invention finds that STING can regulate and control the phosphorylation state of ULK1 in the autophagy process through direct interaction with ULK1, and can influence the proceeding of autophagy flow, so that through protein-protein interaction, STING agonist inhibits ULK1 activation by directly combining with ULK1 and inducing phosphorylation of Ser757 site of ULK1, and further inhibits a classical ULK1 mediated autophagy pathway.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a preparation for regulating ULK1 phosphorylation level in the preparation of a product for inhibiting autophagic flow of immune cells. Background Art

[0002] Cellular senescence is an important factor in many chronic diseases and aging-related pathological conditions. STING (Stimulator of Interferon Genes) may play an important role in the aging process, but its specific mechanism is still unclear. At present, the STING signaling pathway has been widely studied, mainly focusing on its role in immune response and antiviral response. In recent years, studies have found that STING is not only involved in immune response, but also plays an important role in autophagy regulation. STING activation affects cell metabolism and aging process by regulating autophagic flux in cells. For example, some studies have pointed out that the activation of STING can promote the lipidation of LC3 protein by recruiting autophagy-related proteins such as WIPI2, thereby initiating the autophagic process. However, most of these studies focus on the effects of short-term STING activation (within a few hours to 12 hours), and have not explored the long-term effects of STING activation on autophagic flux in depth.

[0003] ULK1 (Unc-51 Like Autophagy Activating Kinase 1) is a key kinase in the initiation of autophagy. Its role is mainly to regulate the initiation of autophagy through the phosphorylation of ULK1. Studies have shown that the phosphorylation of ULK1 is regulated by multiple signaling pathways, including ULK1 phosphorylation activated by cell energy sensors (such as mTORC1) and stress signals. Summary of the invention

[0004] The purpose of the present invention is to provide a new target for preparing products for regulating autophagic flow of immune cells based on the direct interaction between STING and ULK1.

[0005] In order to achieve the above object, the present invention provides the use of a preparation for regulating the phosphorylation level of ULK1 in the preparation of a product for inhibiting the autophagic flow of immune cells, wherein the preparation is a STING agonist.

[0006] In a specific embodiment, the STING agonist is DMXAA.

[0007] In a specific embodiment, the STING agonist DMXAA inhibits the autophagic flux of immune cells by increasing the phosphorylation level of ULK1 at Ser757.

[0008] In a specific embodiment, the product for inhibiting autophagy flux of immune cells is one of a drug for treating immune system diseases, a drug for treating neurodegenerative diseases, a drug for treating cardiovascular aging, and an autophagy inhibitory drug.

[0009] In a specific embodiment, the immune system disease includes autoimmune diseases and senile immune diseases.

[0010] The present invention also provides the use of a preparation for regulating the phosphorylation level of ULK1 in the preparation of a drug, wherein the preparation is a ULK1 agonist or a combination of a STING agonist and a ULK1 agonist, and the drug is one of a drug for treating immune system diseases, a drug for treating neurodegenerative diseases, a drug for treating cardiovascular aging, and a drug for inhibiting autophagy.

[0011] In a specific embodiment, the STING agonist is DMXAA, and the ULK1 agonist is LYN-1604.

[0012] In a specific embodiment, the STING agonist DMXAA is used to increase the phosphorylation level of ULK1 at Ser757 to inhibit autophagic flux.

[0013] In a specific embodiment, the ULK1 agonist LYN-1604 is used to reduce the phosphorylation level of ULK1 at Ser757 to restore autophagic flux.

[0014] In a specific embodiment, the immune system disease includes autoimmune diseases and senile immune diseases.

[0015] The beneficial effects of the present invention include at least:

[0016] 1. The present invention found that STING regulates the phosphorylation state of ULK1 during autophagy by directly interacting with ULK1, thereby affecting the progress of autophagic flux. In this way, through protein-protein interaction, STING agonists inhibit ULK1 activation by directly binding to ULK1 and inducing phosphorylation of its Ser757 site, thereby inhibiting the classic ULK1-mediated autophagy pathway.

[0017] 2. The present invention found that the STING agonist DMXAA can increase the phosphorylation level of ULK1 at Ser757 to inhibit autophagic flux, and the ULK1 agonist LYN-1604 can reduce the phosphorylation level of ULK1 at Ser757 to restore autophagic flux. The interaction between STING and ULK1 revealed by the present invention and its role in autophagy regulation provide new targets and new ideas for the treatment of immune aging, immune disorders and autophagy-related immune system diseases (such as autoimmune diseases, senile immune failure, etc.), the treatment of aging-related diseases (neurodegenerative diseases, cardiovascular aging) and the development of drugs for autophagy inhibition, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Transmission electron microscope images of the control group (Control), the DMXAA group treated for 3 h, and the DMXAA group treated for 72 h in Example 1;

[0019] Figure 2 It is a comparison diagram of the number of autophagosomes in the control group (Control) and the DMXAA group treated for 3 hours and the DMXAA group treated for 72 hours in Example 1;

[0020] Figure 3 The results of the AdPlus-mCherry-GFP-LC3B autophagy flow detection in the control group (Control) and the DMXAA group treated for 3 h and the DMXAA group treated for 72 h in Example 1;

[0021] Figure 4 The Western blot corresponding band diagrams of the autophagy-related proteins Atg5, p62, and LC3B proteins in the control group and the DMXAA group at different time points (2 hours, 4 hours, 24 hours, 48 ​​hours, and 72 hours) in Example 1;

[0022] Figure 5 Western blot corresponding band diagram of p62 and LC3B autophagic fluxes in the control group and the DMXAA group and their respective combined BafA1 treatments in Example 1;

[0023] Figure 6 The corresponding band graphs of Western blot of T-ULK1 and STING in the control group, DMXAA group (3 hours, 48 ​​hours), IgG group immunoprecipitated with non-specific immunoglobulin of the same type as the target antibody, and STING group immunoprecipitated with an antibody specifically targeting the STING protein (3 hours, 48 ​​hours) in Example 1;

[0024] Figure 7The corresponding band diagrams of Western blot of P-ULK1 and T-ULK1 in the control group (Control) and the DMXAA group (treated for 72 hours) in Example 1;

[0025] Figure 8 This is a comparison chart of the phosphorylation levels of ULK1 Ser757 in the control group (Control) and the DMXAA group in Example 1 obtained based on statistical analysis of Western blot results;

[0026] Fig. 9 The immunofluorescence double labeling results of STING and p-ULK1 in the DMXAA group (treated for 72 h) in Example 1, wherein Fig. 9 a in the figure is the immunofluorescence staining result of p-ULK1. Fig. 9 b in the figure is the immunofluorescence staining result of STING. Fig. 9 The c in the figure is the result after staining with Hochest dye. Fig. 9 The d in Fig. 9 The picture after a, b and c are merged;

[0027] Fig.10 Transmission electron microscopy images of the control group (Control), DMXAA group, LYN-1604 group and DMXAA+LYN-1604 group in Example 2;

[0028] Fig.11 This is a comparison chart of the number of autophagosomes in the control group (Control), DMXAA group, LYN-1604 group and DMXAA+LYN-1604 group in Example 2;

[0029] Fig.12 1 and 2. These are the Western blot corresponding band diagrams of p62 and LC3B autophagic fluxes in the control group, DMXAA group, LYN-1604 group, and DMXAA+LYN-1604 group in Example 2 and their respective combined BafA1 treatments. DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to the accompanying drawings and embodiments, but the present invention can be implemented in many different ways as limited and covered by the claims.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Specifically: vascular disruptor DMXAA (5,6-dimethylxanthine-4-acetic acid, Cat#HY-10964) was purchased from MedChemExpress for activating STING; LYN-1604 (Cat#HY-101923) was purchased from MedChemExpress for activating ULK1; lysosomal inhibitor BafA1 (Cat#HY-100558) was purchased from MedChemExpress for inhibiting lysosomal acidification; p-ULK1 (Ser757) antibody (Cat#14202S) was purchased from Cell Signaling Technology; STING antibody (Cat#13647S) was purchased from Cell Signaling Technology; LC3B antibody (Cat#18725-1-AP) was purchased from Proteintech, and P62 antibody (Cat#39749) was purchased from Cell Signaling Technology.

[0032] Example 1

[0033] Cell type: Mouse bone marrow-derived macrophages (BMDM).

[0034] Experimental equipment: laser confocal microscope (Zeiss LSM 880), Western Blot equipment (Bio-Rad, PowerPac Basic) and transmission electron microscope (Hitachi HT7700).

[0035] Extraction and culture of BMDM: bilateral femurs and iliac bones were obtained from C57BL / 6 mice (6-8 weeks old). The bone surface was disinfected with 70% ethanol. The femur and iliac bones were carefully cut open at both ends with surgical scissors and a scalpel, and the bone marrow fluid was flushed out of the bone cavity with PBS. The obtained bone marrow suspension was transferred to a culture bottle containing PBS and filtered using a sterile filter to remove bone fragments and impurities. The cell suspension was centrifuged (500×g, 5 minutes), and after removing the supernatant, it was resuspended in a complete culture medium containing 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution (such as penicillin / streptomycin) and 50ng / mL M-CSF (macrophage colony stimulating factor). The cell suspension was transferred to a culture bottle (a culture bottle suitable for culturing bone marrow-derived macrophages) and cultured in a constant temperature incubator at 37°C and 5% CO2. The cells were cultured at 37°C and 5% CO2 for 6-7 days, and the culture medium was changed every 2-3 days. M-CSF can promote the differentiation of bone marrow cells into macrophages. During the culture process, macrophages will gradually attach to the surface of the culture flask and begin to expand, becoming a typical macrophage morphology.

[0036] BMDM treatment: BMDM to be seeded in six-well plates were induced with M-CSF for 6-7 days to form typical macrophage morphology before drug treatment. The cells were divided into two groups: control group: no treatment; DMXAA group: treated with 50μg / ml DMXAA. Cells in all treatment groups were cultured under the same conditions (37°C, 5% CO2) for 72 hours to observe the effects of treatment on cells.

[0037] 1.1 Observation of autophagosomes using transmission electron microscopy

[0038] In the experiment of observing autophagosomes under transmission electron microscopy, BMDM cells were first extracted and processed, and then cell samples were prepared for observation through steps such as fixation, dehydration, and resin embedding. 2.5% glutaraldehyde was used for fixation, and dehydration was gradually carried out through ethanol solutions of different concentrations. Finally, the cells were immersed and solidified with pure resin. After ultrathin sectioning, the cell sections were stained with heavy metal solutions (such as lead citrate and urea lead) to enhance the contrast. Subsequently, the sections were placed in a transmission electron microscope for observation to evaluate the morphology, number, and distribution of autophagosomes. Finally, the changes in autophagic flow were evaluated by quantitatively analyzing the number of autophagosomes in TEM images to understand the changes in autophagic activity under different treatment conditions.

[0039] Transmission electron microscopy images are shown in Figure 1 .from Figure 1It can be seen that the untreated cells in the control group showed a normal number of autophagic vesicles, indicating that the autophagic flow was not affected; 3 hours after DMXAA treatment, the number of autophagic vesicles in BMDM cells increased significantly; 72 hours after DMXAA treatment, the number of autophagic vesicles in BMDM cells decreased significantly and was lower than the level of the untreated period, indicating that the autophagic flow was inhibited.

[0040] The number of autophagosomes in TEM images was quantitatively analyzed. Specifically, at least 10 cells were counted in each group, and the average number of autophagosomes was calculated. The quantitative analysis results of TEM images are detailed in Figure 2 As shown, from Figure 2 It can be seen that after 72 h of DMXAA treatment, the number of autophagosomes was significantly reduced, indicating that the autophagic flux was inhibited.

[0041] It should be noted that the quantitative analysis data were obtained through quantitative statistics of TEM images.

[0042] 1.2 Autophagic flux detection of AdPlus-mCherry-GFP-LC3B

[0043] AdPlus-mCherry-GFP-LC3B vector was used for fluorescent labeling of autophagosomes and analysis of autophagosome formation. After treatment, laser confocal microscopy was used to observe the number of autophagosomes and their distribution in BMDM cells.

[0044] The results of the AdPlus-mCherry-GFP-LC3B autophagic flux assay are detailed in Figure 3 As shown, Figure 3 The four images in the first row show the autophagy flow detection results of the control group cells under mCherry, GFP, Hochest 33342 and Merge, respectively. Figure 3 The four images in the second row show the autophagy flow detection results of BMDM cells under mCherry, GFP, Hochest33342 and Merge after DMXAA treatment for 3 hours. Figure 3 The four images in the third row show the results of autophagic flux detection of BMDM cells under mCherry, GFP, Hochest 33342 and Merge after DMXAA treatment for 72 hours.

[0045] from Figure 3 It can be seen that the control group showed more autophagic vesicles, indicating normal autophagic flow; 3 hours after DMXAA treatment, the number of autophagic vesicles in BMDM cells increased significantly; after 72 hours of DMXAA treatment, the autophagosomes in BMDM cells decreased significantly, confirming the inhibition of autophagic flow.

[0046] 1.3 Western blot detection

[0047] Western blot detection method: First, BMDM cells were collected and protein was extracted, and the protein concentration was determined using the BCA method. According to the measured protein concentration, 30 μg of protein was taken for Western blot experiments. In the experiment, the protein was first separated by SDS-PAGE gel electrophoresis, and then the separated protein was transferred to the PVDF membrane using the wet transfer method. After transfer, 5% non-fat milk powder was used to block at room temperature for 1 hour to reduce nonspecific binding. Then, the membrane was incubated with the primary antibody (antibodies for LC3B, p62, Atg5 and ULK1 Ser757 phosphorylation site) overnight at 4°C. On the second day, the membrane was washed 3 times with TBST for 10 minutes each time to remove the unbound primary antibody. Then, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 hour, and the membrane was washed again with TBST 3 times. Finally, it was developed by ECL chemiluminescence, and image data was acquired by exposure and scanning with X-ray film. The quantitative analysis of proteins was performed by comparing the gray value of the band with the gray value of the internal reference protein β-actin to calculate the relative expression level of the target protein.

[0048] The experiment included two groups: a control group and a DMXAA group, aiming to compare the effects of DMXAA treatment on autophagy-related proteins (LC3B, p62, Atg5) and the phosphorylation status of ULK1.

[0049] Western blot results are detailed in Figure 4 and Figure 5 As shown, Figure 4 The expression levels of autophagy-related proteins Atg5, p62, and LC3B in DMXAA at different time points (2 hours, 4 hours, 24 hours, 48 ​​hours, and 72 hours). Figure 5 The lysosomal inhibitor BafA1 was added to observe the autophagic flux of p62 and LC3B in DMXAA at 3 and 72 hours. Figure 4 It can be seen that as the DMXAA treatment time increases, the expression of LC3B and Atg5 is downregulated and p62 accumulates at 72 hours, indicating that autophagic flux is inhibited. The above data show that DMXAA treatment changes the expression of these autophagy-related proteins and inhibits autophagy. Figure 5 It can be seen that after 3 hours of DMXAA treatment, the autophagic flux was activated and the lysosomal degradation of p62 and LC3 increased; while after 72 hours of DMXAA treatment, the autophagic flux was inhibited and the lysosomal degradation of p62 and LC3 decreased.

[0050] On this basis, the addition of BafA1 further reflects the changes in autophagic flux. BafA1 was added at a concentration of 200 nM / l at 69 hours after treatment (i.e. 3 hours before sampling), and the autophagic flux of p62 and LC3B was observed by inhibiting the acidification of lysosomes and blocking the fusion of autophagosomes with lysosomes. For details, see Figure 5 ,from Figure 5 It can be seen that 3 hours after DMXAA treatment combined with BafA1 treatment, the degradation of p62 and LC3B to lysosomes increased, indicating that the autophagic flux was accelerated; 72 hours after DMXAA treatment combined with BafA1 treatment, the degradation of p62 and LC3B to lysosomes decreased, indicating that the autophagic flux was blocked.

[0051] 1.4 Analysis of the interaction between STING and ULK1 and its phosphorylation status

[0052] 1) Co-Immunoprecipitation (Co-IP) experiment

[0053] To further confirm the interaction between STING and ULK1, a Co-IP experiment was performed. First, BMDM cells treated with DMXAA for 3 hours and 48 hours were collected, and the cells were lysed using a lysis buffer containing protease inhibitors to obtain cell lysates. Then, anti-STING antibodies were added to the lysate and incubated for 4 hours to promote the binding of antibodies to STING proteins. Next, pre-prepared magnetic protein A / G magnetic beads were added and incubated for another 4 hours. The magnetic beads were able to bind to the antibodies and precipitate the STING-antibody complex. In order to remove non-specifically bound proteins, the magnetic beads were repeatedly washed 3-4 times with a lysis buffer. Then, the STING-antibody-magnetic bead complex was dissociated with a solution containing SDS and heated to remove protein-protein interactions. Finally, the immunoprecipitated samples were analyzed by Western blot technology, and the co-immunoprecipitated ULK1 protein was detected using anti-ULK1 antibodies. The results showed that after DMXAA treatment, the interaction between STING and ULK1 increased significantly, indicating that STING may affect autophagic flow by regulating the activity of ULK1.

[0054] Figure 6 The results of the Co-IP experiment are shown. Figure 6The corresponding band diagrams of Western blot of T-ULK1 and STING in the control group, DMXAA treatment group (3 hours, 48 ​​hours), IgG group using the same type of non-specific immunoglobulin as the target antibody for immunoprecipitation, and STING group using an antibody specifically targeting the STING protein for immunoprecipitation (3 hours, 48 ​​hours); Among them, the Input group: represents all proteins in the cell lysate, which have not been immunoprecipitated, and the IP group: refers to the precipitation of specific target proteins and their interacting proteins by immunoprecipitation technology. Figure 6 It can be seen that after STING was bound to the magnetic beads and incubated with the cell lysate, DMXAA treatment significantly promoted the interaction between STING and ULK1.

[0055] Figure 7 The changes in p-ULK1 levels after 72 hours of DMXAA treatment were shown. Western blot analysis showed that the Ser757 phosphorylation level of ULK1 was significantly increased after 72 hours of DMXAA treatment, suggesting that STING activation may affect autophagy flow by phosphorylating ULK1.

[0056] Figure 8 The results of statistical analysis of the phosphorylation level of ULK1 Ser757 after DMXAA treatment were presented. Statistical analysis further confirmed that the phosphorylation level of ULK1 Ser757 increased significantly after 72 hours of DMXAA treatment, and the differences between different groups were statistically significant.

[0057] 2) Immunofluorescence double labeling experiment

[0058] Immunofluorescence staining was used to analyze the colocalization of STING and p-ULK1. First, anti-STING antibody and anti-p-ULK1 (S757) antibody were used for incubation, then fluorescently labeled secondary antibody was used for detection, and finally the colocalization was observed by laser confocal microscopy. The results of immunofluorescence double labeling are detailed in Fig. 9 , Fig. 9 a in the figure is the immunofluorescence staining result of p-ULK1. Fig. 9 b in the figure is the immunofluorescence staining result of STING. Fig. 9 The c in the figure is the result after staining with Hochest dye. Fig. 9 The d in Fig. 9 The merged picture of a, b and c in Fig. 9 As can be seen in (d), STING and p-ULK1 are co-localized in the cell.

[0059] Example 2

[0060] Effects of the ULK1 agonist LYN-1604 on autophagic flux after STING activation

[0061] Cell type: Mouse bone marrow-derived macrophages (BMDM).

[0062] Experimental equipment: Western Blot equipment (Bio-Rad, PowerPac Basic) and transmission electron microscope (Hitachi HT7700).

[0063] Culture of mouse bone marrow-derived macrophages: This experiment uses bilateral femurs and iliac bones of C57BL / 6 mice (6-8 weeks old) as sources to extract mouse bone marrow cells. First, disinfect the bone surface with 70% ethanol, then carefully cut the femur and iliac bones at both ends with surgical scissors and scalpels, rinse the bone cavity with PBS, and extract the bone marrow fluid. Transfer the obtained bone marrow suspension to a culture bottle containing PBS and filter it through a sterile filter to remove bone fragments and impurities. Next, the cell suspension is centrifuged (500×g, 5 minutes) to remove the supernatant, and then resuspended in a complete culture medium containing 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution (such as penicillin / streptomycin) and 50ng / mL M-CSF (macrophage colony stimulating factor). Then transfer the cell suspension to a culture bottle and culture it in a constant temperature incubator at 37°C and 5% CO2. Culture for 6-7 days, and change the culture medium every 2-3 days. M-CSF can promote the differentiation of bone marrow cells into macrophages, and the cells will gradually adhere to the surface of the culture flask during the culture process, showing typical macrophage morphology.

[0064] Treatment of mouse bone marrow-derived macrophages: BMDM to be inoculated in six-well plates were induced with M-CSF for 6-7 days to form typical macrophage morphology before drug treatment. The experiment was divided into four groups: control group (no treatment), DMXAA group (treated with 50μg / mL DMXAA), LYN-1604 group (treated with 1μM LYN-1604), and DMXAA+LYN-1604 group (simultaneous treatment with 50μg / mL DMXAA and 1μM LYN-1604). Cells in all experimental groups were cultured at 37°C and 5% CO2 for 72 hours to observe the cell morphology, activity and related biological changes after treatment.

[0065] 2.1 Transmission electron microscopy observation of autophagosomes

[0066] In the experiment of observing autophagosomes under transmission electron microscopy, mouse bone marrow-derived macrophages (BMDM) were first extracted and processed, and then cell samples were prepared for observation through steps such as fixation, dehydration, and resin embedding. 2.5% glutaraldehyde was used for cell fixation, and the dehydration process was carried out step by step using ethanol solutions of different concentrations. Finally, the cells were immersed and solidified with pure resin. After ultrathin sectioning, the cell sections were stained with heavy metal solutions (such as lead citrate and urea lead) to enhance the contrast. Subsequently, the sections were placed in a transmission electron microscope for observation to evaluate the morphology, quantity, and distribution of autophagosomes. For detailed experimental results, see Fig.10 As shown, from Fig.10 It can be seen that the untreated cells in the control group showed a normal number of autophagic vesicles, indicating that the autophagic flux was not affected; 72 hours after DMXAA treatment, the number of autophagic vesicles in BMDM cells decreased significantly and was lower than the level of the untreated period, indicating that the autophagic flux was inhibited; 72 hours after LYN-1604 treatment, the number of autophagic vesicles in BMDM cells increased significantly and was higher than the level of the untreated period, and the surface autophagic flux was promoted; 72 hours after DMXAA combined with LYN-1604 treatment, the number of autophagosomes in BMDM cells increased and was higher than the level 72 hours after DMXAA treatment, indicating that LYN-1604 can reverse the inhibition of autophagic flux caused by DMXAA treatment.

[0067] The number of autophagosomes in TEM images was quantitatively analyzed. Specifically, at least 10 cells were counted in each group, and the average number of autophagosomes was calculated. Fig.11 The quantitative analysis results of TEM images were shown, which clearly reflected the changes in the number of autophagosomes in different treatment groups, indicating that the number of autophagosomes increased significantly after LYN-1604 treatment, further reflecting that LYN-1604 can improve the reduction of autophagic flux and promote the recovery of autophagic activity after DMXAA treatment. Compared with the DMXAA treatment group, the LYN-1604 treatment group showed a significantly increased number of autophagosomes, suggesting that LYN-1604 may play its role in promoting autophagy by improving the changes in autophagic flux.

[0068] 2.2 Western blot detection

[0069] After collecting cells and extracting proteins, the protein concentration was determined using a protein quantification kit and Western blot detection was performed. The Western blot detection method is detailed in the Western blot detection method in Section 1.3 of Example 1.

[0070] The experimental grouping of this experiment includes four groups: control group, DMXAA group, LYN-1604 group and DMXAA+LYN-1604 group. The purpose is to analyze the autophagy flux of autophagy-related proteins (LC3B, p62) by Western blot detection, explore the effects of DMXAA, LYN-1604 and their combined treatment on autophagy, and further understand the intervention effect of LYN-1604 on DMXAA-induced autophagy inhibition.

[0071] In this step, the Western blot results are shown in Fig.12 , Fig.12 The Western blot corresponding band diagram of the changes in p62 and LC3B autophagic fluxes in the control group, DMXAA group, LYN-1604 group, and DMXAA+LYN-1604 group and their combined BafA1 treatment. Fig.12 It can be seen that LYN-1604 can effectively restore the number of autophagosomes and promote the expression of LC3B and Atg5, thereby enhancing autophagic flow and supporting the interaction between ULK1 and STING.

[0072] In addition, the addition of BafA1 further reflects the changes in autophagic flux. BafA1 was added at a concentration of 200 nM / l at 69 hours after treatment (i.e., 3 hours before sampling) to further observe the changes in the autophagic flux of p62 and LC3B by inhibiting the acidification of lysosomes and blocking the fusion of autophagosomes with lysosomes. For details, see Fig.12 ,from Fig.12 It can be seen that 72 hours after DMXAA treatment combined with BafA1 treatment, the degradation of p62 and LC3B to lysosomes decreased, indicating that the autophagic flux was blocked; 72 hours after LYN-1604 treatment combined with BafA1 treatment, the degradation of p62 and LC3B to lysosomes increased, indicating that the autophagic flux was accelerated; 72 hours after DMXAA combined with LYN-1604 treatment and combined with BafA1 treatment, the degradation of p62 and LC3B to lysosomes increased, and was higher than the level 72 hours after DMXAA treatment, indicating that LYN-1604 can reverse the inhibition of autophagic flux caused by DMXAA treatment.

[0073] It should be noted that the band diagram obtained by Western blot detection of the present invention ( Figures 4 to 6 and Fig.12 ) in the table, “-” and “+” mean: “-” means unprocessed, “+” means processed, Fig.12 Take this as an example to illustrate: Fig.12The meanings of “-” and “+” corresponding to LYN-1604, DMXAA and BafA1 can be understood as follows: “-” represents untreated, “+” represents treated, the first to fourth columns represent the control group, DMXAA group, LYN-1604 group and DMXAA+LYN-1604 group not treated with BafA1, respectively, and the fifth to eighth columns represent the control group, DMXAA group, LYN-1604 group and DMXAA+LYN-1604 group treated with BafA1, respectively.

[0074] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions and substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. Use of a preparation for regulating ULK1 phosphorylation level in the preparation of a product for inhibiting autophagic flow of immune cells, characterized in that: The formulation is a STING agonist.

2. The use according to claim 1, characterized in that: The STING agonist is DMXAA.

3. The use according to claim 1, characterized in that: The STING agonist DMXAA inhibits the autophagic flux of immune cells by increasing the phosphorylation level of ULK1 at the Ser757 site.

4. The use according to claim 2, characterized in that: The product for inhibiting the autophagy flow of immune cells is one of a drug for treating immune system diseases, a drug for treating neurodegenerative diseases, a drug for treating cardiovascular aging, and an autophagy inhibitory drug.

5. The use according to claim 4, characterized in that: The immune system diseases include autoimmune diseases and senile immune diseases.

6. Use of a preparation for regulating ULK1 phosphorylation level in the preparation of a drug, characterized in that: The preparation is a ULK1 agonist or a combination of a STING agonist and a ULK1 agonist, and the drug is one of a drug for treating immune system diseases, a drug for treating neurodegenerative diseases, a drug for treating cardiovascular aging, and a drug for inhibiting autophagy.

7. The use according to claim 6, characterized in that: The STING agonist is DMXAA, and the ULK1 agonist is LYN-1604.

8. The use according to claim 7, characterized in that: The STING agonist DMXAA was used to increase the phosphorylation level of ULK1 at Ser757 to inhibit autophagic flux.

9. The use according to claim 7, characterized in that: The ULK1 agonist LYN-1604 is used to reduce the phosphorylation level of ULK1 at the Ser757 site to restore autophagic flux.

10. The use according to claim 6, characterized in that: The immune system diseases include autoimmune diseases and senile immune diseases.

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