A method of high-throughput screening for compounds that promote mitochondrial degradation in inflammatory microglia
By using flow cytometry in microglia, combined with 96-well plates and mitochondrial fluorescent probe staining, the low throughput and complex operation of existing screening methods have been solved, achieving efficient, simple, and quantitative compound screening, thus improving its specificity and clinical application potential.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-06-03
- Publication Date
- 2026-07-21
AI Technical Summary
Existing methods for screening those that promote mitochondrial degradation in inflammatory microglia have low throughput, are cumbersome to operate, are difficult to quantify, and rely on complex data models or expensive imaging equipment, making them difficult to promote and apply in routine laboratories.
Using 96-well or 24-well plates combined with flow cytometry, microglia were stimulated with an inflammation inducer, and candidate drugs were added. Mitochondrial-specific fluorescent probe staining was then performed, and flow cytometry was used to detect changes in fluorescence intensity of candidate drugs on microglia mitochondria, thereby screening for compounds that promote mitochondrial degradation.
It has achieved a high-throughput, rapid, objective, and quantitative screening method, which simplifies the operation, reduces costs, and improves the specificity and clinical translation potential of the screening.
Smart Images
Figure CN122428017A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical screening technology, specifically relating to a method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia. Background Technology
[0002] Neuroinflammation is a complex pathological process caused by excessive activation of immune cells in the central nervous system. It is common in various neurological diseases and is characterized by high disability rates and a high disease burden. For example, the global prevalence of Parkinson's disease has exceeded 6 million cases, and its incidence increased 2.5 times from 1990 to 2016. Parkinson's disease, along with other neurodegenerative diseases such as Alzheimer's disease, is closely related to chronic neuroinflammation. Neurological dysfunction caused by excessive activation due to neuroinflammation is a core pathological feature in the progression of many neurological diseases. Inflammatory activation of microglia is a major source of neuroinflammation. Mitochondria, as the cell's energy factories and signaling centers, play a crucial role in maintaining the health of neurons and glial cells through quality control. Mitochondrial damage in microglia leads to the release of damage-associated molecular patterns (DAMPs) such as mitochondrial DNA (mtDNA). Damaged mitochondria release mtDAMPs, primarily including mtDNA, ATP, and reactive oxygen species. These molecules can activate pattern recognition receptors, triggering innate immune signaling pathways such as the NLRP3 inflammasome and cGAS-STING, inducing the cGAS-STING pathway and inflammasome assembly, and further exacerbating neuroinflammation. Microglial inflammatory responses mediated by the mtDNA-cGAS-STING-NLRP3 signaling axis have been confirmed as an important pathological mechanism in various neurological diseases. Therefore, clearing damaged mitochondria from microglia can alleviate neuroinflammation and improve neurological function.
[0003] Currently, traditional methods for screening drugs that promote mitochondrial degradation mainly include Western blot detection of mitochondrial proteins and electron microscopy observation of mitochondrial morphology. These methods have low throughput, are cumbersome to operate, and are difficult to quantify, making them unsuitable for large-scale drug screening. Furthermore, although some studies have established methods for screening mitophagy inducers based on structural virtual screening or AI-assisted imaging systems, these technologies rely on complex data models or expensive imaging equipment, and often require multi-channel fluorescent labeling and specialized image analysis algorithms, limiting their widespread application in routine laboratories. Therefore, establishing a high-throughput, rapid, objective, and quantitative screening method is of significant practical value. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia.
[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia, comprising the following steps: (1) Establishment of a microglia mitochondrial accumulation model: Microglia, selected from the BV2 cell line or primary microglia, were stimulated with an inflammatory inducer to induce an inflammatory activation state accompanied by mitochondrial accumulation. The inflammation inducer is preferably total DNA or lipopolysaccharide (LPS) derived from mouse brain. The concentration of the inflammation inducer is as follows: 25-100 μg / mL for mouse brain DNA (preferably 50 μg / mL); and 0.5-2 μg / mL for LPS (preferably 1 μg / mL).
[0006] (2) Treatment of candidate drugs: The candidate drug to be screened is added to the inflammatory-activated microglia obtained in step (1), and the candidate drug and the inflammatory inducer are co-incubated for 18-26 hours (preferably 24 hours).
[0007] (3) Dye labeling of mitochondria: Collect the cells after step (2) treatment and add mitochondrial-specific fluorescent probes for staining in the dark; The preferred mitochondrial-specific fluorescent probe is MitoTrackerGreen (working concentration preferably 100-300 nM, staining time preferably 15-30 minutes, room temperature, protected from light).
[0008] (4) Flow cytometry detection and data analysis: The cells stained in step (3) were analyzed by flow cytometry to obtain the fluorescence intensity (mean fluorescence intensity, MFI); and negative control group: microglia without any treatment; positive control group: microglia treated only with inflammatory inducers (such as DNA or LPS) without the addition of candidate drugs; the mitochondrial fluorescence intensity of the positive control group was used as the baseline (defined as mitochondrial accumulation status). Screening criteria: If the mean fluorescence intensity of the candidate drug treatment group was significantly reduced compared with the positive control group (P<0.05), it indicates that the candidate drug has the activity of promoting mitochondrial degradation in inflammatory microglia.
[0009] The beneficial effects of this invention are as follows: (1) High throughput: Using 96-well or 24-well plates combined with flow cytometry, dozens or even hundreds of candidate drugs can be screened rapidly at the same time.
[0010] (2) Objective quantification: Flow cytometry can detect the average fluorescence intensity of tens of thousands of cells, and the results are statistically significant, avoiding the subjectivity of microscopic observation.
[0011] (3) Simple operation: No need to lyse cells or perform protein electrophoresis. After staining, the sample can be directly tested on the instrument and the process can be completed in a few minutes.
[0012] (4) High specificity: This invention uses an inflammation inducer to pre-stimulate and establish a mitochondrial accumulation model. The selected candidate drugs directly target "excessive accumulation of mitochondria under pathological conditions", which improves the specificity of the screening and its clinical translation potential.
[0013] (5) Lower cost: Compared with screening methods based on high content imaging, flow cytometer equipment has a high penetration rate and low consumable costs. Attached Figure Description
[0014] Figure 1 To investigate the effect of compound M1-M8 on mitochondrial content in a microglia (BV2) cell line model of mitochondrial accumulation, flow cytometry was used (compared with control group: ***P<0.001, compared with model group: #P<0.05). Figure 2 The effect of compound M4 on the expression of mitochondrial proteins TOM20 and TIM23 in microglia in an inflammatory state (compared with the control group: *P<0.05, **P<0.01; compared with the model group: ##P<0.01, ###P<0.001). Figure 3 To investigate the effect of compound M4 on mitochondrial autophagy in the inflammatory microglial cell line (BV2 cells) using the mt-Keima model (*P<0.05 compared with the DNA model group). Detailed Implementation
[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.
[0016] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.
[0017] Example 1: Flow cytometry detection of the effects of compounds M1-M8 on mitochondrial content in microglia Experimental principle: MitoTrackerGreen is a mitochondrial-targeting dye. Its chloromethyl group covalently binds to the thiol groups of mitochondrial proteins, and upon excitation, it emits green fluorescence in the mitochondrial lipid environment. The fluorescence intensity of MitoTrackerGreen directly reflects the mitochondrial content.
[0018] The specific implementation steps were as follows: Mouse microglia (BV2 cells, purchased from Yuanjing Biotechnology, catalog number YC-C035) were seeded at 1×10⁵ cells / well in 12-well plates and allowed to grow to a density of 60–70%. 15 μM compounds M1–M8 (dissolved in DMSO, a compound that may promote mitochondrial degradation, patent pending) were co-treated with 50 μg / mL total DNA derived from mouse brain (total DNA extracted from the cerebral cortex of C57BL / 6 mice using an RNA / DNA extraction kit manufactured by Beyotime, catalog number R0017S) for 24 hours. The drug-containing culture medium was discarded, and the cells were washed twice with PBS. BV2 cells were resuspended in 200 nM MitoTrackerGreen (purchased from Beyotime, catalog number C1048) HBSS solution and incubated at room temperature in the dark for 15 min. Subsequently, the cells were centrifuged at 800×g for 5 min at room temperature, resuspended in 150 μL PBS, and the process was repeated once. Cell suspensions were filtered through a 70 μm filter and then analyzed using a flow cytometer with FlowJo V10 software. The fluorescence intensity of the control group (veh, treated with DMSO solvent) was set to 0%, and the fluorescence intensity of the model group (veh+DNA, treated with DMSO solvent and 50 μg / mL total DNA derived from mouse brain) was set to 100%. The relative fluorescence intensity of each treatment group was then calculated.
[0019] The results are as follows Figure 1 As shown, compared with the control group (veh), the model group (veh+DNA) showed a significant increase in MitoTrackerGreen fluorescence intensity after treatment with total DNA derived from mouse brain, indicating that total DNA derived from mouse brain induced an increase in mitochondrial content in BV2 cells. Compared with the model group (veh+DNA), treatment with M2 and M4 significantly reduced the fluorescence intensity of MitoTrackerGreen in BV2 cells, indicating that M2 and M4 have the effect of reducing mitochondrial content in microglia.
[0020] Example 2: Detection of the effect of compound M4 on mitochondrial proteins using Western blotting To verify that the molecules screened in Example 1 that promote mitochondrial degradation in microglia can reduce mitochondrial content at the protein level, we used different concentrations of M4 to treat microglia in a model of mitochondrial accumulation induced by total DNA from mouse brain. We used Western blotting to detect constitutively expressed mitochondrial proteins TIM23 and TOM20, whose expression levels can reflect mitochondrial content.
[0021] The specific implementation steps were as follows: BV2 cells were co-treated with different concentrations of compound M4 and total DNA derived from mouse brain for 24 hours, and the cell pellets from each group were collected. Total protein was extracted and quantified using the BCA method. An equal amount of protein (30 μg) was subjected to SDS-PAGE electrophoresis, transferred to a membrane, blocked, and incubated overnight at 4°C with primary antibodies TIM23 (Abclonal, catalog number A27746), TOM20 (Abclonal, catalog number A19403), and β-actin (Abclonal, catalog number AC026). After washing, the membrane was incubated at room temperature for 2 hours with HRP-labeled secondary antibody (Abclonal, catalog number AS014), and finally analyzed by ECL chemiluminescence immunoassay.
[0022] like Figure 2 The results showed that, compared with the control group, the expression of mitochondrial proteins TIM23 and TOM20 was significantly increased after treatment in the model group, suggesting that DNA induces the accumulation of mitochondrial content in BV2 cells. Compared with the model group, 15 μM of compound M4 significantly reduced the expression of TIM23 and TOM20, suggesting that compound M4 can reduce the mitochondrial content in microglia.
[0023] Example 3: Detection of the effect of compound M4 on mitochondrial protein degradation using mt-Keima To verify the molecule screened in Example 1 that promotes mitochondrial degradation in microglia, we further used mt-Keima imaging to detect its degradative effect on microglia mitochondria. mt-Keima is a pH-sensitive fluorescent protein derived from coral that, after modification with a mitochondrial matrix targeting signal, can specifically locate in the mitochondrial matrix. This protein emits green fluorescence when excited at 488 nm in a neutral environment (normal mitochondria, pH 7.4); and emits red fluorescence when excited at 555 nm in an acidic environment (lysosomes, pH 4.5), with the intensity of red fluorescence positively correlated with the level of mitophagy. Therefore, if compound treatment leads to more mitochondria in an acidic environment (i.e., promoting mitophagy), it indicates that the compound has the effect of reducing mitochondrial content in cells.
[0024] mt-KeimamRNA (purchased from GenScript) was transfected into BV2 cells (using Lipofectamine 3000 transfection, purchased from Thermo Fisher). After culturing for 4 hours to induce protein expression, control groups (Ctrl, equal volume of DMSO solvent), positive control groups (Rapa, 500 nM Papamycin, which significantly promotes mitophagy and reduces mitochondrial content, purchased from MCE), DNA groups (50 μg / mL total DNA derived from mouse brain), and DNA+M4 groups (50 μg / mL total DNA derived from mouse brain + 15 μM M4) were set up. After co-incubation for 24 hours, live cell images were taken under a confocal microscope. The emission light at 600-650 nm was excited at 488 nm and collected, defined as green pseudocolor; the emission light at 600-650 nm was excited at 555 nm and collected, defined as red pseudocolor. In a neutral environment, mitochondria exhibited yellow fluorescence (a superposition of green and red pseudocolor); in an acidic environment, mitochondria exhibited red fluorescence. The red fluorescent spots represent mitochondria that have entered the lysosomes. The more numerous the spots, the more mitochondria are being degraded in the cell, ultimately leading to a decrease in mitochondrial content.
[0025] like Figure 3 As shown (left image is a typical confocal plot, right image is a semi-quantitative statistical plot), the number of red fluorescent dots per unit cell was counted, with each data point representing one cell. Compared with the control group, the positive control group (Rapa) significantly increased the number of red fluorescent dots per unit cell, indicating that the constructed mt-Keima model can effectively detect compounds that promote mitochondrial degradation. Compared with the DNA group, the DNA+M4 group showed a significant increase in red dot structures (DNA group: 0.73±0.80 vs. DNA+M4 group: 2.13±1.55, P=0.019, data are expressed as mean ± standard deviation), suggesting that mitochondria enter lysosomes for degradation. These results indicate that compound M4 can promote mitophagy in inflammatory microglia and has the effect of reducing cellular mitochondrial content. Figure 3 The results showed that the compounds screened by the flow cytometry system in Example 1 also had the effect of reducing mitochondrial content in the mt-Keima model.
[0026] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.
Claims
1. A method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia, characterized in that, Includes the following steps: (1) Stimulate microglia with inflammatory inducers to establish a microglia mitochondrial accumulation model; (2) Add the candidate drug to be screened and the inflammation inducer described in step (1) together into microglia and incubate them together; (3) Collect the cells treated in step (2) and add mitochondrial-specific fluorescent probes for staining in the dark; (4) Detect the cells stained in step (3) by flow cytometry to obtain the average fluorescence intensity of the cells. Set up a negative control group and a positive control group. Use the mitochondrial fluorescence intensity of the positive control group as the baseline. If the average fluorescence intensity of the candidate drug treatment group is significantly lower than that of the positive control group, it indicates that the candidate drug has the activity of promoting the degradation of mitochondria in inflammatory microglia.
2. The method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia according to claim 1, characterized in that, The microglia mentioned in step (1) are selected from the BV2 cell line or primary microglia.
3. The method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia according to claim 1, characterized in that, The inflammation inducer mentioned in step (1) is total DNA or lipopolysaccharide (LPS) derived from mouse brain.
4. The method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia according to claim 3, characterized in that, The total DNA derived from the mouse brain was used at a concentration of 25 μg / mL to 100 μg / mL.
5. The method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia according to claim 3, characterized in that, The concentration of LPS used is 0.5 μg / mL to 2 μg / mL.
6. The method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia according to claim 1, characterized in that, The co-incubation time in step (2) is 18 to 26 hours.
7. The method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia according to claim 1, characterized in that, The mitochondrial-specific fluorescent probe mentioned in step (3) is MitoTrackerGreen.
8. The method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia according to claim 7, characterized in that, The working concentration of MitoTrackerGreen is 100nM to 300nM, the staining time is 15 minutes to 30 minutes, and the staining conditions are room temperature and protection from light.
9. The method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia according to claim 1, characterized in that, The negative control group in step (4) consists of untreated microglia, and the positive control group consists of microglia treated only with an inflammatory inducer without the addition of the candidate drug.
10. The method for high-throughput screening of compounds that promote mitochondrial degradation in inflammatory microglia according to claim 1, characterized in that, The significant reduction mentioned in step (4) refers to P < 0.05.