Medicine for reversing oligodendrocyte apoptosis based on lysosome function recovery and application

Abstract

The invention provides a medicine for reversing oligodendrocyte apoptosis based on lysosome function recovery and application, and belongs to the technical field of biological medicine. The invention proves that autophagic flow retardation and endoplasmic reticulum stress induced by neutrophil extracellular trapping nets (NETs) can be effectively reversed by recovering lysosome functions, and the apoptosis rate of oligodendrocytes and the level of reactive oxygen species (ROS) are remarkably reduced, so that the survival state of the cells is improved. Experimental data show that the lysosome activator EN6 can promote generation of autophagy lysosome, accelerate degradation of an autophagy substrate and cooperatively inhibit expression of endoplasmic reticulum stress key protein. The scheme provided by the invention provides a new target for treatment of secondary brain injury after cerebral hemorrhage, can be expanded to intervention strategies of other NETs related diseases, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to drugs for reversing oligodendrocyte apoptosis based on restoring lysosomal function and their applications. Background Art

[0002] Intracerebral Hemorrhage (ICH) is a severe subtype of hemorrhagic stroke, with high disability and mortality rates. Its pathological mechanism is complex. In addition to the mechanical injury of the primary hematoma, secondary brain injury involves processes such as inflammatory response, oxidative stress, and abnormal activation of immune cells. Studies have shown that neutrophils infiltrate massively in the local microenvironment after intracerebral hemorrhage and participate in secondary injury by releasing Neutrophil Extracellular Traps (NETs). NETs are composed of degranulated chromatin DNA, histones, and antibacterial proteins, and were initially considered an important immune defense mechanism for clearing pathogens. However, under non-infectious pathological conditions, such as intracerebral hemorrhage, the excessive release of NETs may exacerbate tissue damage, but its specific mechanism of action has not been fully elucidated.

[0003] Oligodendrocytes, as key supporting cells in the central nervous system, are responsible for myelin formation and axon protection. Their functional impairment is closely related to neurodegenerative diseases and neurological dysfunction after acute brain injury. Existing studies have found that inflammatory factors, oxidative stress, and abnormal immune responses in the microenvironment after intracerebral hemorrhage can induce oligodendrocyte apoptosis, but the specific regulatory pathway of NETs in this process is still unclear. Currently, there is no effective means to improve cell survival by restoring lysosomal function or regulating autophagy flux for NETs-induced oligodendrocyte apoptosis. Summary of the Invention

[0004] The purpose of the present invention is to provide drugs for reversing oligodendrocyte apoptosis based on restoring lysosomal function and their applications, providing an effective means for the research of related diseases.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides the application of lysosome activators and / or endoplasmic reticulum stress inhibitors in the preparation of drugs for reducing oligodendrocyte damage or apoptosis based on restoring autophagy flux activity.

[0007] Preferably, the lysosome activator is EN6.

[0008] The present invention provides the application of drugs for restoring autophagy flux activity in the preparation of drugs for preventing and / or treating secondary brain injury after intracerebral hemorrhage.

[0009] The present invention provides the use of an endoplasmic reticulum stress inhibitor and a lysosome activator in the co - preparation of a drug for preventing and / or treating secondary brain injury after intracerebral hemorrhage.

[0010] Preferably, the endoplasmic reticulum stress inhibitor is 4 - PBA.

[0011] The present invention provides an in vitro model for evaluating the efficacy of a drug on secondary brain injury after intracerebral hemorrhage, characterized in that the model uses the mCherry - GFP - LC3B dual - fluorescence system to trace the autophagic flux activity.

[0012] The present invention provides a pharmaceutical composition for preventing and / or treating secondary brain injury after intracerebral hemorrhage, characterized by comprising a lysosome activator and an endoplasmic reticulum stress inhibitor.

[0013] Preferably, the lysosome activator is EN6; the endoplasmic reticulum stress inhibitor is 4 - PBA.

[0014] The present invention provides an oligodendrocyte protector based on the restoration of autophagic flux, and the protector is achieved by activating the lysosome function.

[0015] The present invention provides a model for in vitro simulating NETs - induced oligodendrocyte apoptosis, and detects autophagic flux arrest and endoplasmic reticulum stress - related indexes after NETs intervention.

[0016] Advantages of the present invention:

[0017] The present invention confirms that by restoring the lysosome function, it effectively reverses the autophagic flux arrest and endoplasmic reticulum stress induced by neutrophil extracellular traps (NETs), significantly reduces the apoptosis rate of oligodendrocytes and the level of reactive oxygen species (ROS), thereby improving the cell survival state. Experimental data show that the lysosome activator EN6 can promote the generation of autophagolysosomes, accelerate the degradation of autophagic substrates, and synergistically inhibit the expression of key proteins of endoplasmic reticulum stress. The solution provided by the present invention provides a new target for the treatment of secondary brain injury after intracerebral hemorrhage, and can also be extended to the intervention strategies for other NETs - related diseases, with broad clinical application prospects. Description of the Drawings

[0018] Figure 1To investigate the effect of autophagy-endoplasmic reticulum stress regulation on the survival of oligodendrocytes, where Figure A shows representative images of Calcein-AM / PI double staining; B shows quantitative analysis of PI-positive cells (n = 3); C shows detection of cell viability by CCK-8 (n = 3); D shows DCFH-DA staining to indicate ROS levels; E shows quantification of ROS fluorescence intensity (n = 3). Scale bar of fluorescence images = 500 μm; "#" indicates comparison with the Blank group, #P < 0.05, ##P < 0.01, P < 0.001, #P < 0.0001; "*" indicates comparison between groups, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001;

[0019] Figure 2 To provide morphological evidence for lysosomal function reconstruction, where Figure A shows autophagolysosome structures revealed by transmission electron microscopy; B shows dual fluorescence tracing of mCherry-GFP-LC3B; C shows autophagic flux index (mCherry / GFP ratio, n = 3); D shows LysoTrackerRed staining; E shows average fluorescence intensity of LysoTrackerRed (n = 3). Scale bar of large-field TEM images = 5 μm, scale bar of local magnified immunofluorescence TEM images of cells = 1 μm; scale bar of dual fluorescence system tracing images of mCherry-GFP-LC3B = 20 μm;

[0020] Scale bar of Lyso-TrackerRed staining images = 200 μm; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001;

[0021] Figure 3 To investigate the effect of autophagic flux inhibition and endoplasmic reticulum stress on apoptosis, where Figure A shows fluorescence images of Annexin V-FITC / PI double staining; B shows the number of late apoptotic cells (n = 3). Scale bar of AnnexinV-FITC / PI double staining fluorescence images = 200 μm; "#" indicates comparison with the Blank group, #P < 0.05,

[0022] ##P < 0.01, P < 0.001, #P < 0.0001; "*" indicates comparison between groups, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001;

[0023] Figure 4To investigate the effects of restored autophagic flux on apoptosis and endoplasmic reticulum (ER) stress. A. Western blot analysis of the protein expression levels of LC3, p62, GRP78, and cleaved caspase-3. B-E. Semi-quantitative analysis of protein expression (n = 3). "#" indicates comparison with the Blank group, #P<0.05, ##P<0.01,

[0024] P<0.001, #P<0.0001; "*" indicates comparison between groups, *P<0.05, **P<0.01,

[0025] ***P<0.001, ****P<0.001. Detailed implementation manners

[0026] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] Embodiment

[0028] Since both ER stress and autophagy play important roles in maintaining cellular homeostasis and responding to damage, the present invention explores whether inhibiting ER stress and restoring autophagic flux can improve the survival status of oligodendrocytes and compares the differences in their cytoprotective effects.

[0029] After NETs intervention, cells were treated with the lysosomal inhibitor Baf-A1, the lysosomal activator EN6, and the ER stress inhibitor 4-PBA, respectively, and the cell survival status was evaluated. The results of the cell viability and death staining assay showed that compared with the NETs group, the number of PI-positive cells in the Baf-A1 treatment group was significantly increased (P<0.0001, Figure 1 A and B), indicating a further increase in cell mortality. Meanwhile, the cell viability in the Baf-A1 treatment group was significantly decreased (P<0.0001, Figure 1 C), and the intracellular ROS level was significantly increased (P<0.0001, Figure 1 D and E). In contrast, the treatments with the lysosomal activator EN6 and the ER stress inhibitor 4-PBA both significantly improved cell survival. In the EN6 and 4-PBA treatment groups, the proportion of PI-positive cells was significantly decreased compared with the NETs group (P<0.001 vs. EN6, P<0.01 vs. 4-PBA, Figure 1 B), the cell viability was significantly enhanced (P<0.001 vs. EN6, P<0.05 vs. 4-PBA, Figure 1 C), and the intracellular ROS level was significantly decreased (P<0.0001 vs. EN6, P<0.0001 vs. 4-PBA, Figure 1D and E). Notably, among all the detected indices, the protective effect of the EN6 group was significantly better than that of the 4-PBA group (the number of PI-positive cells decreased, P < 0.05; cell viability increased, P < 0.05; ROS level decreased, P < 0.01).

[0030] The restoration of autophagic flux may play a key role in cell survival and in response to NETs-induced damage. To explore the effects of the regulation of autophagic flux on apoptosis and endoplasmic reticulum stress, we used TEM and the mCherry-GFP-LC3B dual-fluorescent labeling system to analyze the restoration of autophagic flux in detail. The TEM results showed that compared with the NETs group, the number of autolysosomes in cells increased significantly after treatment with the lysosome activator EN6 ( Figure 2 A). Through tracing with the mCherry-GFP-LC3B dual-fluorescent system ( Figure 2 B), it was found that compared with the NETs group, autophagic flux was significantly restored after treatment with the lysosome activator EN6, further verifying this phenomenon (P < 0.0001, Figure 2 C), and its autophagic flux activity even exceeded that of the blank group (P < 0.05, Figure 2 C). The fluorescence intensity of Lyso-TrackerRed in the EN6 group was restored and was comparable to that of the blank group ( Figure 2 D and E).

[0031] To evaluate the effects of autophagic flux inhibition and endoplasmic reticulum stress on apoptosis, we treated cells with Baf-A1, EN6, and 4-PBA respectively after NETs intervention, and quantitatively analyzed apoptosis by AnnexinV / PI double staining ( Figure 3 A). The results showed that the proportion of late apoptotic cells in the Baf-A1 treatment group was significantly higher than that in the NETs group (P < 0.0001, Figure 3 B). While the EN6 treatment group could effectively reduce the proportion of late apoptotic cells (P < 0.0001) and restore it to the level of the blank group. Although in comparison, 4-PBA could also reduce the apoptosis rate (P < 0.001), its protective effect was significantly weaker than that of EN6 (P < 0.05).

[0032] Western blot analysis verified the changes in proteins related to autophagy, endoplasmic reticulum stress, and apoptosis ( Figure 4 A). After treatment with Baf-A1, the expression level of the autophagy marker LC3-II reached a peak (P < 0.001 vs. blank, Figure 4B), indicating a large accumulation of autophagosomes that cannot be degraded, while after EN6 treatment, the LC3-II level was significantly reduced to the control group level (P = nsvs.blank, P < 0.05 vs. NETs). The expression of LC3-II in the 4-PBA group also decreased (P < 0.05 vs. NETs group), but the degradation efficiency seemed to be lower than that of the EN6 group. In addition, the degradation efficiency of the autophagy substrate p62 was significantly higher in the EN6 group than in the 4-PBA group (P < 0.05, Figure 4 C). In terms of endoplasmic reticulum stress regulation, both EN6 and 4-PBA significantly reduced the expression of GRP78, and there was no significant difference between them ( Figure 4 D). In addition, EN6 was more significant in reducing the expression of cleaved-caspase3, and the effect was better than that of the 4-PBA group (P < 0.01, Figure 4 E).

[0033] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a lysosome activator and / or an endoplasmic reticulum stress inhibitor in the preparation of a medicament for reducing oligodendrocyte damage or apoptosis based on restoring autophagic flux activity.

2. The application according to claim 1, characterized in that, The lysosome activator is EN6.

3. Use of a medicament for restoring autophagic flux activity in the preparation of a medicament for preventing and / or treating secondary brain injury after intracerebral hemorrhage.

4. Use of an endoplasmic reticulum stress inhibitor and a lysosome activator in the co-preparation of a medicament for preventing and / or treating secondary brain injury after intracerebral hemorrhage.

5. The application according to claim 1, characterized in that, The endoplasmic reticulum stress inhibitor is 4-PBA.

6. An in vitro model for evaluating the efficacy of drugs on secondary brain injury after intracerebral hemorrhage, characterized in that, The model uses the mCherry-GFP-LC3B dual-fluorescence system to trace autophagic flux activity.

7. A pharmaceutical composition for preventing and / or treating secondary brain injury after cerebral hemorrhage, characterized in that, It contains a lysosome activator and an endoplasmic reticulum stress inhibitor.

8. The pharmaceutical composition according to claim 7, wherein The lysosome activator is EN6; the endoplasmic reticulum stress inhibitor is 4-PBA.

9. An oligodendrocyte protector based on the restoration of autophagic flux, characterized in that, The protective agent is achieved by activating lysosome function.

10. An in vitro model for simulating NETs-induced apoptosis of oligodendrocytes, characterized in that, After NETs intervention, autophagic flux blockade and endoplasmic reticulum stress-related indicators are detected.

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