Use of cryab in medical diagnosis and therapy

Abstract

The present application discloses the use of alphaB-crystallin (CRYAB) as a marker for diagnosing or predicting a seizure disorder, particularly epilepsy. The present application also discloses the use of alphaB-crystallin in the preparation of a medicament or a kit for preventing or treating a seizure disorder, particularly epilepsy.

Description

Technical Field

[0001] This invention relates to the biomedical field, specifically to the use of αB-crystallin (CRYAB) as a biomarker in the diagnosis of paroxysmal encephalopathy, especially epilepsy, and also to the use of CRYAB in the preparation of drugs or kits for the prevention or treatment of paroxysmal encephalopathy, especially epilepsy. Background Technology

[0002] Epilepsy is a clinical syndrome characterized by recurrent epileptic seizures caused by abnormal, synchronized discharges of neurons in the brain. Globally, approximately 65 million people suffer from epilepsy, and it has been listed by the World Health Organization as one of the five most important neuropsychiatric disorders requiring global prevention and control (Asadi-Pooya, 2023). Most epilepsy patients can control their seizures with antiepileptic drugs, but 30% of patients do not respond to medication. A deeper understanding of its etiology and pathogenesis is urgently needed to develop new antiepileptic drugs and treatments. Traditionally, epilepsy was considered a gray matter disorder or neuronal disorder. A recent breakthrough is the recognition that epilepsy is also a white matter disorder or myelin disorder (Hogan, 2020). Driven by neuronal activity, the myelin structure of the myelin sheath changes according to neuronal activity, a process called adaptive myelination. In epileptic states, repeated abnormal neuronal activity leads to increased myelin content and myelin formation in nerve fibers, a phenomenon known as maladaptive myelination. The relationship between myelin plasticity and disease (epilepsy) is considered a frontier in neuroscience and clinical neurology research for the next decade (Bonetto, 2021; Xin, 2020). αB-crystallin (CRYAB) is a small chaperone protein of the heat shock protein family (Hayashi, 2020) and is highly expressed in oligodendrocytes (OLs) that form myelin sheaths.

[0003] Existing technologies mainly involve the diagnostic and therapeutic effects of CRYAB in autoimmune diseases, especially multiple sclerosis. The biggest difference between these technologies and this application is that they do not involve paroxysmal encephalopathy, especially epilepsy. Currently, there is no application of CRYAB in the diagnosis and treatment of paroxysmal encephalopathy, especially epilepsy. Summary of the Invention

[0004] The first aspect of the present invention provides the use of CRYAB or a CRYAB detection reagent.

[0005] Specifically, the use of CRYAB or a CRYAB detection reagent in the preparation of reagents or kits for diagnosing paroxysmal encephalopathy is provided, wherein the amino acid sequence of CRYAB is shown in SEQ ID NO: 1.

[0006] The use of CRYAB or a CRYAB detection reagent in the preparation of reagents or kits for predicting the onset of paroxysmal encephalopathy is also provided, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] In a preferred embodiment, the above-described uses further include analyzing the expression and / or protein levels of CRYAB or CRYAB in biological samples selected from blood, serum, plasma, saliva, cerebrospinal fluid, urine, feces, and tissue samples.

[0008] In a preferred embodiment, the above-described uses further include detecting the expression of CRYAB or CRYAB in oligodendrocytes or exosomes derived from oligodendrocytes.

[0009] A second aspect of the present invention provides the use of CRYAB, CRYAB derivatives or CRYAB exosomes in the preparation of medicaments or kits for the prevention, relief or treatment of paroxysmal encephalopathy, wherein the amino acid sequence of CRYAB is shown in SEQ ID NO: 1.

[0010] The CRYAB derivatives therein comprise conjugates of CRYAB and / or pharmaceutically acceptable salts of CRYAB.

[0011] A third aspect of the invention provides a method for treating paroxysmal encephalopathy.

[0012] In a preferred embodiment, a therapeutically effective dose of CRYAB is administered to a patient via parenteral administration, wherein the dose is effective in inhibiting the progression of an established disease.

[0013] In a preferred embodiment, the pharmaceutical composition is administered to an individual via intra-arterial, intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, or oral administration.

[0014] In a preferred embodiment, the administration includes the following steps: administering to the patient an effective amount of the conjugate and / or a mixture containing a pharmaceutically acceptable carrier.

[0015] The paroxysmal encephalopathy described in this invention refers to a brain disease characterized by paroxysmal episodes, often exhibiting features such as sudden onset, recurrence, and self-limitation, manifesting as epileptic seizures and / or non-epileptic seizures.

[0016] The paroxysmal encephalopathy described in this invention is preferably epilepsy.

[0017] In this invention, paroxysmal encephalopathy / paroxysmal neurological disorders refer to epilepsy, paroxysmal disorientation, stupor, etc.

[0018] The terms "episodic encephalopathy" and "episodic brain disease" are used interchangeably. They refer to clinical brain diseases characterized by sudden onset, recurrence, and self-limitation, including epileptic encephalopathy (DEE) and recurrent encephalopathy.

[0019] The CRYAB or CRYAB detection reagents described in this invention include CRYAB-specific antibodies and detection methods such as high-resolution mass spectrometry.

[0020] Technical effect

[0021] The inventors discovered that CRYAB can serve as a pathological marker for epileptogenic foci, primarily expressed in oligodendrocytes. In the cortical tissue of epilepsy patients, increased CRYAB levels are accompanied by elevated expression of inflammatory factors. In the plasma of epilepsy patients, oligodendrocyte-derived exosomal CRYAB expression is elevated. Therefore, CRYAB can serve as a diagnostic marker for paroxysmal disorders, especially epilepsy.

[0022] The inventors' research found that kaempferol (KA) administration to the corpus callosum and cortex of epileptic mice led to increased expression of CRYAB-positive fibers, and that CRYAB exerts its anti-epileptic effect by inhibiting inflammatory factors. The inventors' research suggests that CRYAB can be used to prepare therapeutic drugs for paroxysmal disorders, especially epilepsy. Attached Figure Description

[0023] Figure 1 A paraffin section of human brain tissue stained with Laufer Quick Blue, the white matter portion of the temporal lobe cortex of an epileptic patient; Figure 1 Comparison of LFB optical density values ​​between the epilepsy group and the control group; Figure 1 c Immunohistochemical staining of paraffin sections of human brain tissue, temporal lobe white matter of an epileptic patient; Figure 1 Comparison of CRYAB-positive cells between the epilepsy group and the control group; Figure 1 Western blotting gel image of e-crystal protein; Figure 1 Comparison of crystal protein expression levels between the epilepsy group and the control group;

[0024] Figure 2 To identify oligodendrocyte lineages expressing CRYAB using different markers. Figure 2 a represents the Olig 2 marker; Figure 2 b indicates the use of SOX10 markers; Figure 2 c indicates the use of NG2 markers; Figure 2 d represents the use of GFAP markers; Figure 2 e indicates that the Olig 1 marker is used; Figure 2 f indicates the use of Olig 3 markers; Figure 2 g represents Olig SP markers; Figure 2 h represents the Iba1 marker; Figure 2 i indicates the use of CRYAB markers; Figure 2 j represents the NeuN marker; Figure 2 k is a common marker for both CRYAB and NeuN; Figure 2 m represents the CRYAB marker; Figure 2 n represents the use of PV markers; Figure 2 o is a common marker for both CRYAB and PV; Figure 2 l and Figure 2 p represents Figure 2 k and Figure 2 The enlarged image within the white box in o. Among them, Figure 2 The scale bars for a, b, c, and d are 10 μm. Figure 2 The scale bars for e, f, g, and h are 50 μm; Figure 2 The scale bars for i, j, and k are 100 μm; Figure 2 The scale bars for m, n, and o are 100 μm. Figure 2 The l and p scales are 50 μm.

[0025] Figure 3 a) Western Blot analysis of CRYAB expression, an inflammatory factor, in temporal lobe cortex tissue; Figure 3 b is a statistical graph of CRYAB expression of inflammatory factor. * indicates P<0.05, ** indicates p<0.01, and **** indicates p<0.001.

[0026] Figure 4 a represents the expression of related proteins in human brain tissue, human plasma, and plasma exosomes; Figure 4 b represents the expression of marker proteins from different cell sources, and the proteins were purified using antibodies from different cell sources; Figure 4 c shows the morphological and structural features of purified exosomes as revealed by transmission electron microscopy. Figure 4 d shows that the purified exosomes have a particle size distribution between 50-200 nm, as indicated by exosome nanotracing (NTA) technology. Figure 4 e represents the expression of CRYAB and CD81 in exosomes from different sources; Figure 4 f represents the expression levels of CRYAB and CD81 in plasma exosomes of the control group and epilepsy patients; Figure 4 g represents the comparison of CRYAB expression levels in plasma exosomes of the control group and epilepsy patients; ** indicates p<0.01.

[0027] Figure 5 a represents mouse behavior at different rating levels recorded by a three-dimensional behavioral video monitoring system; Figure 5b is the original EEG recording of the mouse at the time corresponding to Figure a; Figure 5 c is a screenshot from the video recording of the behavior;

[0028] Figure 5 d represents CRYAB immunofluorescence staining of the corpus callosum and cortex of mice after KA modeling; Figure 5 e represents the number of cells in the hippocampus co-expressing CRYAB, olig2, and Iba1.

[0029] Figure 6 6a shows the changes in seizure frequency and related protein expression levels in a mouse epilepsy model after CRYAB administration. 6a is a statistical graph of the number of grand mal seizures in mice in different co-treatment groups; 6b is an immunoblot plot of each inflammatory factor in different treatment groups; 6c is a statistical graph of the relative expression levels of each inflammatory factor in different treatment groups.

[0030] Figure 7 To demonstrate the effect of chemogenetic-specific upregulation of Cryab expression, the image shows a stained brain slice from a Cryab-cre mouse injected with hM3Dq-mCherry virus into the hippocampus. Figure 7 a represents the control group, which uses Cryab labeled with fluorescein 488; Figure 7 b represents the spontaneous mCherry signal in the control group; Figure 7 c is Figure 7 a magnified view of a section; Figure 7 d is Figure 7 b. Enlarged view of a specific area; Figure 7 e represents the CNO-activated group, which uses Cryab labeled with fluorescein 488; Figure 7 f represents the spontaneous mCherry signal of the CNO activation group; Figure 7 g is Figure 7 e. Enlarged view of a specific area; Figure 7 h is Figure 7 f. Enlarged view of a specific area; Figure 7 i represents the control group, which uses fluorescein 488 to label Cryab and fluorescein 647 to label c-fos and DAPI signals. Figure 7 j is Figure 7 Enlarged view of a portion of the i-Hippocampus CA1 region; Figure 7 k is Figure 7 Enlarged view of a portion of the i-Hippocampus CA3 region; Figure 7 l is Figure 7 Enlarged view of the DG area of ​​the i-Hippocampus; Figure 7 m represents the CNO-activated group, which uses fluorescein 488 to label Cryab, fluorescein 647 to label c-fos and DAPI signals; Figure 7 n is Figure 7 Enlarged view of a portion of the CA1 region of the hippocampus; Figure 7 o for Figure 7 Enlarged view of a portion of the CA3 region of the hippocampus; Figure 7 p is Figure 7 Enlarged view of a portion of the DG region of the hippocampus; Figure 7 The scale bars for a, b, e, f, i, and m are 800 μm. Figure 7 The scale bars for c, d, g, h, j, k, l, n, o, and p are 80 μm.

[0031] Figure 8 The graph shows the ROC and DCA analysis of CRYAB concentration. A represents the ROC curve of CRYAB concentration in diagnosing gliomas with and without preoperative epilepsy. Figure 8 B is the ROC curve of CRYAB concentration in the diagnosis of epilepsy; Figure 8 C represents the ROC curve of CRYAB concentration in the diagnosis of refractory epilepsy and epilepsy; Figure 8 D represents the decision curve of plasma CRYAB circulating levels for gliomas with and without preoperative epilepsy. Figure 8 E represents the decision curve of plasma CRYAB circulating levels for epileptic patients and healthy controls; Figure 8 F represents the decision curve of plasma CRYAB circulating levels for refractory epilepsy and epilepsy. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0033] Unless otherwise specified, all materials and reagents described in the following examples are commercially available.

[0034] In the following embodiments, the experimental methods such as Western blot detection and flow cytometry are conventional experimental methods well known to those skilled in the art. Where no specific conditions are specified in the experimental methods, they are usually operated according to conventional conditions.

[0035] The human brain tissue sections described in the following examples were obtained from volunteer donations from the Chinese Brain Tissue Resource Bank. Plasma from epilepsy patients was obtained from clinical research samples from Huashan Hospital affiliated with Fudan University. Plasma from glioma patients was obtained from clinical research samples from Huashan Hospital affiliated with Fudan University. Temporal lobe cortex tissue was obtained from clinical research samples from Huashan Hospital affiliated with Fudan University.

[0036] Example 1: Staining of paraffin sections of human brain tissue using the Laufer Quick Blue staining method

[0037] The paraffin sections of human brain tissue were stained using the Laufer Quick Blue staining method, and the steps are as follows:

[0038] 1. Dewaxing the sections to water: (xylene → graded alcohol → distilled water), soak in xylene I and II for 10 min each, soak in 100% alcohol I and II → 95% alcohol → 90% alcohol → 80% alcohol → 70% alcohol for 5 min each, and soak in distilled water for 5 min;

[0039] 2. Place the tissue sections in Luxol Fast Blue (LFB) staining solution and incubate overnight at room temperature;

[0040] 3. Wash with 95% ethanol until no blue particles remain on the tissue surface;

[0041] 4. Rinse with deionized water for 5 minutes;

[0042] 5.0.05% lithium carbonate was differentiated for 10 seconds (dispersion needs to be observed under a light microscope) until the gray and white matter were clearly distinguishable;

[0043] 6. Rinse with deionized water for 5 minutes;

[0044] 7. For re-dyeing with tar purple dye, place the dye vat in a 37℃ constant temperature oven for 10 minutes;

[0045] 8. Rinse with deionized water for 5 minutes;

[0046] 9. Differentiate in tar purple solution for 5 seconds (observation under a light microscope is required during differentiation);

[0047] 10. Rinse with deionized water for 5 minutes;

[0048] 11. Dehydration and transparency: (gradient alcohol → xylene) Soak in 70% alcohol → 80% alcohol → 90% alcohol for 5 seconds each, soak in 100% alcohol I and II for 3 minutes each, and soak in xylene I and II for 10 minutes each;

[0049] Precautions: After each soaking step, lift the slicer up and down several times before transferring it to the next liquid for better results;

[0050] 12. Neutral resin mounting: wet mounting (lower the fume hood glass to arm height for protection) drop a small amount of resin onto the tissue, cover with a coverslip, place in the fume hood and wait for the resin to dry. It can then be observed under a light microscope and stored at room temperature.

[0051] Lauffer fast blue stained paraffin sections of human brain tissue, such as Figure 1 As shown in figure a, myelin staining in the temporal lobe cortex white matter of epilepsy patients was significantly increased. Compared with the healthy control group (n=10), the LFB optical density (OD) value was significantly increased in the epilepsy group (n=9), p<0.001. Figure 1 b).

[0052] Example 2: Immunohistochemical staining of crystal proteins in paraffin sections of human brain tissue

[0053] The paraffin sections of human brain tissue were stained using crystalloid protein immunohistochemistry, and the steps are as follows:

[0054] 1. Dewaxing the sections to water: (xylene → graded alcohol → distilled water), soak in xylene I and II for 10 min each, soak in 100% alcohol I and II → 95% alcohol → 90% alcohol → 80% alcohol → 70% alcohol for 5 min each, and soak in distilled water for 5 min;

[0055] Precautions: After each soaking step, lift the slicer up and down several times before transferring it to the next liquid for better results;

[0056] 2. Transfer the slides to a glass staining jar and add PBS. Place the jar on a shaker and shake slowly. Wash the slides three times with PBS buffer, 10 minutes each time.

[0057] 3. Antigen retrieval: The sections were retrievald in an antigen retrieval pressure cooker using TrisHCl pH9.0 buffer. After retrieval, the sections were allowed to cool to room temperature before being removed.

[0058] 4. Place the extracted slides into a glass staining jar and add PBS. Place the jar on a shaker and shake slowly. Wash with PBS buffer three times, 10 minutes each time.

[0059] 5. Permeability Adjustment: (This solution should be prepared fresh and protected from light during preparation.) First, prepare 0.3% Triton solution with PBS, then dissolve it thoroughly in a 37°C water bath by stirring. Before use, add 0.3% hydrogen peroxide and mix thoroughly. Pour the permeability adjustment solution into a staining jar containing glass slides and incubate at 37°C in the dark for 30 minutes to increase the permeability of the slides.

[0060] 6. Wash three times with PBS buffer, 10 minutes each time (shake gently on a shaker);

[0061] 7. Blocking: Reagent preparation: 5% donkey serum was prepared with PBST;

[0062] Procedure: Remove the slide, blot the water around the tissue with filter paper, draw a circle around the tissue with an immunohistochemical pen, place it in a humidified chamber, and drop about 150 μL of donkey serum into the circle (to completely cover the tissue). Then place the humidified chamber in a constant temperature water bath (37°C) for 30 minutes.

[0063] 8. Incubation of Antibody I: Preparation of reagents: Prepare the antibody (Anti-Alpha B Crystallin antibody [1B6.1-3G4], Mouse, ab13496, Abcam) with PBST at a ratio of 1:500 to prepare the working solution of Antibody I;

[0064] Procedure: Remove the humidified chamber, gently remove the donkey serum from the slides with a pipette, add about 150 μL of I-antibody working solution (enough to completely cover the tissue), and place the slides back into the humidified chamber in a constant temperature water bath (37°C) for 60 min.

[0065] 9. Remove the wet box from the water bath and place it in a 4°C refrigerator overnight (more than 16 hours);

[0066] 10. Remove the humidifier box from the refrigerator and let it sit at room temperature for 60 minutes to allow it to return to room temperature;

[0067] 11. Discard the antibody, place the slide in a staining jar, and wash it three times with PBS, 10 minutes each time;

[0068] 12. Incubation of Antibody II: Preparation of reagents: Prepare Antibody II (Horse Anti-Mouse IgG Antibody (H+L), Biotinylated, BA-2000, VECTOR) at a ratio of 1:200 PBST to make Antibody II working solution; Procedure: After washing the slide, use filter paper to absorb the water around the circle, add about 150 μL of Antibody II working solution (to completely cover the tissue), place it in a humidified box and put it in a constant temperature water bath (37°C) for 120 minutes;

[0069] 13. Remove the humidified chamber, discard the working solution of antibody II, place the slide in the staining jar and wash it three times with PBS, 10 minutes each time;

[0070] 14. Incubation with ABC solution: Use filter paper to absorb the water around the circle of the slide, add about 150ul of ABC solution (ABC-HRP Kit, PK-4000, VECTOR) (1:200, prepared 30 minutes in advance) with a pipette to completely cover the tissue, and place the humidified box in a constant temperature water bath (37℃) for 60 minutes.

[0071] 15. Remove the humidified chamber, place the slides in the staining jar, and wash three times with PBS, 10 minutes each time;

[0072] 16. Color development: Quickly add DAB working solution (50ul of 1% DAB; 1ul of 30% hydrogen peroxide plus PBST to 1ml, DAB (D5637, SIGMA)), observe the staining, and quickly discard the staining solution;

[0073] Precautions: ① DAB is toxic by contact. Be sure to wear latex gloves and handle with care during this step. ② First, observe each slide with the naked eye. If any slides turn yellow, quickly place them under a microscope to confirm a positive result (the background just turns yellow and the positive result appears as a sesame-like brown color). Terminate the reaction with PBS. The maximum color development time is 15 minutes.

[0074] 17. Place the slide in a staining jar and wash with PBS for 10 minutes, then wash with double-distilled water for 10 minutes;

[0075] 18. Dehydration and transparency: (gradient alcohol → xylene) Soak in 70% alcohol → 80% alcohol → 90% alcohol → 100% alcohol I and II for 5 minutes each, and soak in xylene I and II for 10 minutes each.

[0076] Precautions: After each soaking step, lift the slicer up and down several times before transferring it into the next liquid for better results;

[0077] 19. Neutral resin mounting: wet mounting (lower the fume hood glass to arm height for protection) drop a small amount of resin onto the tissue, cover with a coverslip, place in the fume hood and wait for the resin to dry. After drying, it can be observed under a light microscope and stored at room temperature.

[0078] Immunohistochemical staining of paraffin sections of human brain tissue, such as... Figure 1 As shown in c, CRYAB is selectively expressed in white matter. Myelin staining was significantly increased in the temporal lobe cortex white matter of epilepsy patients. Compared with the healthy control group (n=27), the epilepsy group (n=18) showed a significant increase in CRYAB-positive cells (*p<0.01). Figure 1 d).

[0079] Example 3: Human brain tissue crystal protein-Western Blot experiment

[0080] SDS-PAGE adhesive is prepared according to the following table 1:

[0081] Table 1

[0082]

[0083]

[0084] Sample loading: Add 5 µL of pre-stained protein marker (WJ101, Yaxin) to the second well of each gel, and add 10 µL of tissue protein to each of the remaining wells in sequence. (Note: It is best not to use the first and last wells of each gel to obtain better protein bands.)

[0085] SDS-PAGE electrophoresis was performed at a constant voltage of 120V for 70 minutes. (Note: The electrophoresis time depends on the position of the pre-stained marker bands and the size of the target protein. The molecular weight of the crystalline protein in this experiment was approximately 25 kDa.)

[0086] Cut a PVDF membrane (0.45µm, IPVH00010, Millipore) the same size as the gel, mark one end with a pen, soak it in methanol for about 5 minutes, and then soak it in transfer buffer. At the same time, soak the cut gel, filter paper and sponge used in the transfer in the transfer buffer. Then stack the membrane and gel in the following order: black multiwell plate - sponge - filter paper - gel - membrane - sponge - white multiwell plate, close and clamp them, and place them in the transfer tank in the direction of "black to black, white to red". Add an ice box, pour in the transfer buffer, and place the transfer tank in ice to keep it at a low temperature.

[0087] Transfer: 280mA constant current transfer for 120 minutes.

[0088] After the transfer is complete, remove the membrane, stain it with 1x Ponceau S for about 1 minute, then rinse it with ddH2O until bands are visible, and observe the transfer process.

[0089] The membrane containing the target protein was placed in a blocking solution and sealed, and then shaken at room temperature for 60 minutes.

[0090] Incubation of Antibody I: Antibody (Anti-Alpha B Crystallin antibody [1B6.1-3G4], Mouse, ab13496, Abcam) was prepared at a ratio of 1:1000 with 0.5% TBST buffer, and internal control (GAPDH) was prepared at a ratio of 1:2000 with 0.5% TBST buffer to prepare Antibody I working solution. The membrane was placed in a humidified chamber, and the working solution was slowly and evenly spread on the membrane with a pipette. The humidified chamber was then placed in a 4°C refrigerator overnight.

[0091] The next day, remove the humidifier and leave it at room temperature for 60 minutes.

[0092] TBS-T wash mask, 3 times for 15 minutes each time.

[0093] Secondary antibody incubation: Prepare the working solution of secondary antibody (Anti-Mouse IgG (H+L), HRP Conjugate, W4021, Promega) at a ratio of 1:2000 with TBS-T at room temperature. Place the membrane in a humidified chamber and slowly and evenly cover the membrane with the working solution using a pipette. Then place the humidified chamber at room temperature for 120 minutes.

[0094] TBS-T wash mask, 3 times for 15 minutes each time.

[0095] ECL Development: ECL is prepared by mixing two reagents (enhanced luminescence solution A, BL520B-1; stabilizer solution B, BL520B-2, Biosharp) in a 1:1 ratio. Prepare the solution immediately before use. When drawing the two reagents, use separate pipette tips to avoid mixing them. Avoid direct sunlight during incubation. Develop the solution using a Western blotting (WB) developer and save the image.

[0096] Western blotting further demonstrated that, compared with the healthy control group, CRYAB expression was significantly increased in the temporal lobe white matter of the epilepsy group, such as... Figure 1 As shown in e and f.

[0097] Example 4: Increased CRYAB expression accompanied by increased expression of inflammatory factors

[0098] Using the Western blotting method described in Example 3, the expression of inflammatory cytokine proteins and CRYAB in temporal lobe cortex tissue was detected. The control group consisted of healthy individuals (n=14), and the epilepsy group consisted of patients with epilepsy (n=16). The results showed that compared with the control group, the expression of CRYAB in the temporal lobe cortex tissue of the epilepsy group was significantly increased, accompanied by increased expression of inflammatory cytokines IL-1β, TNFα, NFκB, and pNFκB. Figure 3 As shown in a and b.

[0099] Example 5: Markers for oligodendrocyte lineages

[0100] To further identify oligodendrocytes expressing CRYAB ( Figure 2 (Medium green) The markers of oligodendrocyte lineages were systematically observed. Olig 1, Olig 2, Olig 3 and OligSP ( Figure 2 (In a, e, f, g, the red ones) are transcription factors essential for oligodendrocyte development; SOX10 ( Figure 2 (b in red) is a transcription factor that guides neural stem cells to develop into glial cells; NG2 ( Figure 2 CRYAB (c-red) is a membrane-bound chondroitin sulfate proteoglycan expressed by oligodendrocyte precursor cells and is now used as one of the markers for oligodendrocyte precursor cells. CRYAB coexists with multiple markers derived from oligodendrocyte lineages, allowing for the combined use of various markers to identify oligodendrocytes. Figure 2 ah).

[0101] Astrocyte marker GFAP ( Figure 2 d (red) and the marker of microglia Iba1 ( Figure 2 No co-expression of CRAYB (red) and CRYAB was observed in the cell bodies. CRAYB (green) and the neuronal marker NeuN (red) were also observed. Figure 2 i, j, k, l) and the marker PV (red) of interneurons ( Figure 2 m, n, o, p) are also not expressed together.

[0102] Example 6: Preparation of CRYAB exosomes and their nano-tracing and Western Blot detection of CRYAB expression in exosomes

[0103] (1) Preparation of exosomes

[0104] 1. Take 200 μL of plasma sample from an epilepsy patient and thaw it at room temperature until it is completely liquid, then place it on ice for later use.

[0105] 2. Centrifuge the plasma sample at 2000x g for 20 min at 4°C to remove cells and debris, and transfer the clear supernatant into a new tube.

[0106] 3. Centrifuge the sample at 10000x g for 20 min at 4°C, and transfer the clear supernatant into a new tube and place it on ice for later use.

[0107] 4. Add 100 μL of PBS to 200 μL of clear plasma and mix by blowing and aspiration. Add 60 μL of coprecipitation reagent to the mixture. Mix by blowing and aspiration, and incubate at room temperature for 10 minutes. Centrifuge the sample at 10000 x g for 5 minutes at room temperature. Discard the supernatant; the precipitate is total exosomes.

[0108] 5. Add 200 μL of PBS to a new tube. Add 0.4 μL of CD81 antibody (Santa, sc-7637) to the tube to prepare the CD81 antibody working solution. Add 1 μL of biotinylated antibody (VECTOR, BA-2000) to the CD81 antibody working solution and incubate at room temperature for 2 hours to prepare the biotinylated antibody working solution. Add 10 μL of resin to the incubated biotinylated antibody working solution and incubate at room temperature on a rotary mixer for 1 hour to prepare the resin antibody working solution.

[0109] 6. To enrich neuronal exosomes (NDE), astrocyte-derived exosomes (ADE), microglia-derived exosomes (MDE), and oligodendrocyte-derived exosomes (OED), biotinylated antibody working solutions were prepared using different antibodies and methods. EAAT2 antibody (Abcam, ab41621) was used to enrich ADE. TMEM119 antibody (Biolegend, 853302) was used to enrich MDE. MOG antibody (Abcam, ab243034) was used to enrich OED. For NDE enrichment, 0.4 μL of CD171 antibody (Invitrogen, 13-1719-82) (a commercially available biotinylated antibody) was used. Doses were based on 200 μL of antibody working solution, and antibody culture conditions were as described in Example (1) 5 above.

[0110] 7. Mix the resin antibody working solution thoroughly, and transfer 200 μL to a test tube containing the exosome precipitate; mix well by pipetting. Incubate overnight at 4°C on a rotary mixer. Centrifuge the sample at 2500 x g for 3 min at 4°C, discard the supernatant, and then resuspend in 200 μL of PBS. Repeat twice. Centrifuge the sample at 2500 x g for 3 min at 4°C, and discard the supernatant.

[0111] Subsequently, different resuspension methods were used for subsequent experiments: ① Exosomes were collected for nano-tracing and electron microscopy observation, resuspended in 45 μL of 1M Tris-HCl (pH=8), and incubated at room temperature for 5 min. 5 μL of 50 mM Gly-HCl (pH=3) was added and elution was stopped. The mixture was centrifuged at 2500 g for 3 min at 4 °C, and the supernatant was transferred to a new centrifuge tube; ② Protein solutions were prepared for Western blotting, with 50 μL of RIPA lysis buffer added, and lysed at 4 °C for 1 h for protein concentration determination.

[0112] (2) Electron microscopic observation of exosomes

[0113] 1. Place the carbon nanotube support membrane in a hydrophilometer and ionize it for 10 seconds.

[0114] 2. Use self-locking pointed tweezers to pick up the carbon nanotube support membrane with the carbon membrane side facing up, add 2.5 μL of exosome suspension, and allow it to adsorb for 90 seconds.

[0115] 3. Use filter paper to absorb excess liquid, immediately add an appropriate amount of 2% uranium acetate, then use filter paper to absorb it again. Repeat twice.

[0116] 4. Add 2% uranium acetate to the carbon film and allow it to adsorb for 90 seconds.

[0117] 5. Use filter paper to absorb the liquid, place the sample under a heat lamp to dry, and complete the preparation of the transmission electron microscope sample.

[0118] 6. Exosomes were observed using a TECNAI T12 120KV transmission electron microscope. The purified exosomes were shown to have a 50-200nm circular monolayer membrane structure. The results are as follows: Figure 4 As shown in c.

[0119] (3) Exosome nano-tracing

[0120] 1. Collect 1 mL of the exosome suspension obtained in (1).

[0121] 2. Nanosight 300 nanoparticle tracking analyzer was used for nanoparticle tracking detection, showing that the purified exosomes had a particle size distribution between 50-200 nm, which is consistent with the exosome particle size range. Figure 4 As shown in d.

[0122] (4) Western Blot detection of CRYAB expression in exosomes

[0123] 1. Add BCA reagent (Pierce) TM BCA Protein Assay Kits, 23225, Thermo Scientific TM Mix solutions A, B, and C in a ratio of 25:24:1 to prepare BCA working solution.

[0124] 2. Add 20 μL of protein solution or standard, 80 μL of PBS, and 100 μL of BCA working solution to a 96-well plate, incubate at 37°C for 30 min, and measure the absorption at 595 nm using a microplate reader.

[0125] 3. Convert the sample concentration. Using the lowest concentration sample as the standard, dilute each sample to the same concentration using RIPA lysis buffer (RIPA lysis buffer (medium), P0013C, Beyotime). Add an equal volume of 2X loading buffer and incubate in a boiling water bath for 6 minutes.

[0126] 4. Prepare an SDS-PAGE plate with 10% separating gel + 5% stacking gel, load 10 μL of sample into each well, and perform electrophoresis at 120V for 80 min.

[0127] 5. Using a 0.44μm PVDF membrane, perform electrophoresis at a constant current of 280mA for 2 hours in a buffer solution containing 20% ​​methanol.

[0128] 6.5% skim milk powder sealed at room temperature for 1 hour.

[0129] 7. Incubate different antibodies in different bands overnight at 4°C. Wash the bands three times with TBST for 10 minutes each time.

[0130] 8. Incubate with HRP-labeled secondary antibodies (Anti-Mouse IgG(H+L), HRP Conjugate, W402B, PROMEGA) / (Anti-Rabbit IgG(H+L), HRP Conjugate, W401B, PROMEGA), 1:2000, at room temperature for 2 hours. Wash the bands three times with TBST for 10 minutes each time.

[0131] 9. Expose and develop colors, and calculate the gray values ​​of each band and the relative expression of the protein.

[0132] 10. The band exposure results showed that the concentrations of other neuronal markers in exosomes from a single source were low, and this method can isolate exosomes from a single source.

[0133] By comparing grayscale values ​​and calculating relative protein expression, the results showed that this experiment obtained a sufficient quantity and purity of exosomes. Figure 4 a) Exosomes derived from oligodendrocytes in the plasma of epilepsy patients have a high level of CRYAB expression ( Figure 4 b); CRYAB is most abundantly expressed in exosomes derived from oligodendrocytes and microglia. Figure 4 e); Compared with the control group (n=6), the plasma exosome CRYAB level of epilepsy patients (n=6) was significantly increased (Figures f and g).

[0134] Example 7: Preparation of a mouse model of epilepsy treated with CRYAB

[0135] Kamarinic acid (KA) is known to be widely used to induce acute brain epilepsy seizures and is often used to construct mouse epilepsy models.

[0136] 1. Grouping of the CRYAB treatment experiment for epilepsy. The experiment consisted of three groups: the CRYAB treatment group, the KA treatment group, and the control group. In both the CRYAB treatment group and the KA treatment group, epilepsy was induced by intraperitoneal injection of KA. The CRYAB treatment group received a pre-injection of CRYAB before the KA injection, while the KA treatment group received a pre-injection of normal saline before the KA injection. The control group received two intraperitoneal injections of normal saline.

[0137] 2. Male C57BL / 6 mice (Speford Biotechnology Co., Ltd.) aged 8 weeks were used in the experiment. One week before the modeling began, electrode sockets were installed on the mice. The electrode sockets were welded with 4 screws and fixed with dental cement.

[0138] 3. Place the mice in an EEG observation cage or a 3D behavior recorder box, connect a camera to ensure clear and complete recording of mouse behavior, and use a three-dimensional behavior video monitoring system (animal behavior acquisition and analysis system (3D) BA-DC01, Shenzhen Yiwan Life Technology Co., Ltd.) to record mouse seizures and score them according to the Racine classification.

[0139] 4. Mice in the CRYAB treatment group were injected intraperitoneally with 1 mg / kg CRYAB, while mice in the KA treatment group and the control group were injected with an equal volume of physiological saline. The behavior of the mice was observed and recorded for 30 minutes.

[0140] 5. Mice in the CRYAB treatment group and KA treatment group were injected intraperitoneally with 25 mg / kg KA, while the control group was injected with an equal volume of saline. The epileptic behavior of the mice was observed and recorded for 120 min.

[0141] 6. After recording, the mice were housed alone. 72 hours later, the mice were anesthetized by injecting 80 mg / kg sodium pentobarbital, perfused with PBS and PFA and the whole brain was removed, or fresh brain tissue was taken after anesthesia.

[0142] 7. Record the epileptic state of each mouse, categorized into grades 1 to 5: 1. Rigidity; 2. Continuous head nodding; 3. Twitching of the upper body and forelimbs; 4. Generalized convulsions, standing; 5. Generalized convulsions, loss of balance, jumping, supine. Epileptic behaviors of grade 4 or higher are classified as grand mal seizures. Record the number and duration of grand mal seizures for each mouse, and record the duration and frequency of epileptic waves observed in the computer. Calculate the average value for each group as the standard for judging the severity of the epileptic seizure.

[0143] 8. Compared with the KA treatment group, the CRYAB treatment group showed a significant reduction in the number of grade 4 or higher grand mal seizures, indicating that pre-injection of CRYAB had an anti-epileptic effect in this acute epilepsy model.

[0144] The video monitoring system records the behavior of mice, such as Figure 5 As shown in a and c, the original EEG recordings of mice with epileptic status scores of 2, 3, 4, and 5 are as follows. Figure 5 As shown in b; behavioral video recordings show that the mouse's facial clonic movements, head nodding, and unilateral clonic movements are grade 2 / 3 epileptic seizures. Note that the head has implanted EEG recording electrodes. Figure 5 As shown in c.

[0145] 9. After KA administration to establish a mouse model of epilepsy, immunofluorescence staining was performed, followed by image analysis. Immunofluorescence staining (green) of the corpus callosum and cortex of mice after KA modeling showed an increase in CRYAB-positive nerve fiber markers compared to the control group. Figure 5 As shown in d; where A1-A3, B1-B3, and C1-C3 are high-power images of the areas shown in the box in the left image; statistical analysis showed an increase in CRYAB, olig2, and Iba1 co-expressing cells in the hippocampus of epileptic mice, such as Figure 5 As shown in e.

[0146] Example 8: Western Blot detection of inflammatory factor levels in mouse brain tissue samples

[0147] 1. Take mouse hippocampal tissue frozen at -80℃, add RIPA lysis buffer at a ratio of 250μL per 10mg of tissue, grind the tissue twice at 65Hz for 30s, and let it stand at 4℃ for 1h.

[0148] 2. Centrifuge the sample at 10000x g for 5 min at 4℃, and take the clear supernatant into a new tube for protein concentration determination.

[0149] 3. Prepare BCA working solution by mixing BCA reagent solutions A, B, and C in a ratio of 25:24:1.

[0150] 4. Add 20 μL of protein solution or standard, 80 μL of PBS, and 100 μL of BCA working solution to a 96-well plate, incubate at 37°C for 30 min, and measure the absorption at 595 nm using a microplate reader.

[0151] 5. Convert the sample concentration. Using the lowest concentration sample as the standard, dilute each sample to the same concentration using RIPA lysis buffer. Add an equal volume of 2X loading buffer and incubate in a boiling water bath for 6 minutes.

[0152] 6. Prepare an SDS-PAGE plate with 10% separating gel + 5% stacking gel, load 10 μL of sample into each well, and perform electrophoresis at 120V for 80 min.

[0153] 7. Using a PVDF membrane with a specification of 0.44 μm, perform electrophoresis at a constant current of 280 mA for 2 h in a buffer solution containing 20% ​​methanol.

[0154] 8.5% skim milk powder sealed at room temperature for 1 hour.

[0155] 9. Incubate different inflammatory factor antibodies in different bands overnight at 4°C. Wash the bands three times with TBST for 10 minutes each time.

[0156] 10. Incubate with HRP-labeled secondary antibody, 1:2000, at room temperature for 2 hours. Wash the bands three times with TBST, 10 minutes each time.

[0157] 11. Expose and develop colors, and calculate the gray values ​​of each band and the relative expression of the protein.

[0158] 12. Band exposure results showed that the levels of some inflammatory factors were reduced in the mouse tissue samples of the CRYAB treatment group, suggesting that pre-injection of CRYAB may play a role in reducing inflammation levels during KA-induced epilepsy.

[0159] The results showed that administration of CRYAB to epileptic mice significantly suppressed KA-induced epileptic seizure behavior. Figure 6 a); Immunoblotting shows the expression of representative inflammatory factor proteins ( Figure 6 b); Statistical analysis showed that CRYAB administration significantly reduced the expression of KA-induced inflammatory factors (NF-κB, TNF-α, TLR4). Figure 6 c). Therefore, it can be demonstrated that CRAYB exerts its anti-epileptic effect by intervening in inflammatory cytokine signaling pathways.

[0160] Example 9: Increased cellular expression of CRYAB induced by chemogenetic stimulation

[0161] hM3Dq, modified from the human muscarinic acetylcholine receptor M3 (hM3D), is a DREADD artificial receptor that responds only to clozapine-N-oxide (CNO) and is no longer activated by acetylcholine. CNO acts on cells expressing hM3Dq, causing depolarization and enhancing cellular excitability. In some neurons, CNO binds to hM3D, activating the Gq protein-coupled phosphatase PLCβ, leading to the degradation of phosphatidylinositol 4,5-bisphosphate (PIP2). This opens the KCNQ outward potassium ion channel, which was previously closed by PIP2, resulting in cell membrane depolarization and the formation of an action potential. Cryab-cre mice were constructed, and the constructed pAAV-hSyn-DIO-hM3Dq-mCherry virus was injected bilaterally into the hippocampus of these mice. hSyn is a neuron-specific promoter; the DIO sequence allows cre-positive cells to express the target gene; and mCherry is a commonly used red fluorescent protein. In this system, Cryab exhibits cell-specific expression of hM3Dq, which is specifically activated upon CNO injection. Chemogeneous stimulation increases CRYAB expression in cells. Figure 7 ah showed that after CNO injection to activate hM3Dq, CRYAB cell expression was significantly increased in the whole hippocampus. Figure 7 Following CNO injection to activate hM3Dq, 40 μm frozen sections of brain tissue from experimental mice were prepared. CRYAB and c-fos (a protein expressed early in the brain, characterizing changes in neuronal activity) were fluorescently stained, with saline as a control. Results showed significantly increased expression of CRYAB and c-fos in the CA1, CA3, and DG regions of the hippocampus. Figure 7 ).

[0162] Example 10: ROC and DCA analysis of CRYAB as a diagnostic biomarker

[0163] 1. CRYAB testing in patients with glioma and epilepsy

[0164] Plasma was collected from patients with glioma (including those with and without preoperative epilepsy), patients with epilepsy, healthy controls, and patients with refractory epilepsy. The circulating level of CRYAB in the plasma was measured using the method described in Example 6.

[0165] 2. ROC curve analysis and decision curve analysis

[0166] Figure 8 Receiver operating characteristic (ROC) curve analysis of A showed an AUC value of 0.9135 > 0.9, indicating high diagnostic accuracy. Therefore, CRYAB concentration has strong diagnostic ability in differentiating gliomas with and without preoperative epilepsy. Figure 8ROC curve analysis of B showed that the AUC value was 0.9474 > 0.9, indicating that the diagnostic accuracy was high. Therefore, CRYAB concentration has a strong diagnostic ability in distinguishing epilepsy from healthy controls. Figure 8 ROC curve analysis of C showed that the AUC value was 0.5, indicating that this diagnosis had no diagnostic value. Therefore, CRYAB concentration had no diagnostic ability in distinguishing between refractory epilepsy and epilepsy controls.

[0167] Figure 8 Decision curve analysis (DCA) of D showed clinical benefit in predicting glioma patients with or without preoperative epilepsy by reducing plasma circulating CRYAB levels. Therefore, reducing plasma circulating CRYAB levels provides a larger net benefit in clinical decision-making regarding the prediction of glioma with preoperative epilepsy. Figure 8 E's decision curve analysis showed the clinical benefit of reduced plasma CRYAB circulating levels in predicting epilepsy; therefore, reduced circulating CRYAB levels provide a significant net benefit in clinical decision-making regarding epilepsy prediction. Figure 8 The decision curve for F shows that circulating plasma CRYAB levels do not have a net benefit in predicting refractory epilepsy.

[0168] References

[0169] 1. Asadi-Pooya AA, Brigo F, Lattanzi S, Blumcke I (2023) Adultepilepsy. Lancet 402:412–424.

[0170] 2. Hogan RE(2020) Epilepsy as a Disease of White Matter. Epilepsy Curr21:27–29.

[0171] 3. Bonetto G, Belin D, Káradóttir RT (2021) Myelin: A gatekeeper of activity-dependent circuit plasticity? Science 374:eaba6905.

[0172] 4. Xin W, Chan JR (2020) Myelin plasticity: sculpting circuits in learning and memory. NatRev Neurosci 21:682–694.

[0173] 5.Hayashi J,Carver JA(2020)The multifaceted nature ofαB-crystallin.Cell Stress Chaperones 25:639–654.

[0174] Entertainment

[0175] SEQ ID NO: 1CRYAB promoter

[0176] RRPFFPFHSP SRLFDQFFGE HLLEDLFPT STSLSPFYLR

[0177] PPSFLRAPSW FDTGLSEMRL EKDRFSVNLD VKHFSPEELK VKVLGDVIEV

[0178] HGKHEERQDE HGFISREFHR KYRIPADVDP LTITSSLSSD GVLTVNGPRK

[0179] QVSGPERTIP ITREEKPAVT APPLICATION ID NO:2CRYAB CLASSIFICATION

[0180] aggaggagga ggcaccaaac ggcctgggtg gatagaaggg ggacaaggag gcacacccag

[0181] gccggcaaag agcagctcag tgagtactgg gtatgtgtca cattgccaaa tcccggatca

[0182] caagtctcca tgaactgctg gtgagctagg fatheraaac ccctgacatc accattccag

[0183] aagcttcaca agactgcata fatherggggc tggctgtagc tgcagctgaa ggagctgacc

[0184] agccagctga cccctcacac tcacctagcc acctggaca tcgccatcca ccacccctgg

[0185] atccgccgcc ccttctttcc tttccactcc cccagccgcc tctttgacca gttcttcgga

[0186] gagcacctgt tggagtctga tcttttcccg acgtctactt ccctgagtcc cttctacctt

[0187] cggccaccct ccttcctgcg ggcacccagc tggtttgaca ctggactctc agagatgcgc

[0188] ctggagaagg acaggttctc tgtcaacctg gatgtgaagc acttctcccc agaggaactc

[0189] aaagttaagg tgttgggaga tgtgattgag gtgcatggaa aacatgaaga gcgccaggat

[0190] gaacatggtt tcatctccag ggagttccac aggaaatacc ggatcccagc tgatgtagac

[0191] cctctcacca ttacttcatc cctgtcatct gatggggtcc tcactgtgaa tggaccaagg

[0192] aaacaggtct ctggccctga gcgcaccatt cccatcaccc gtgaagagaa gcctgctgtc

[0193] accgcagccc ccaagaaata gatgcccttt cttgaattgc attttttaaa acaagaaagt

[0194] ttccccacca gtgaatgaaa gtcttgtgac tagtgctgaa gcttattaat gctaagggca

[0195] ggcccaaatt atcaagctaa taaaatatca ttcagcaaca gataaaaaaa aaaaaaaaaa

[0196] aaaa

[0197] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of CRYAB, a CRYAB derivative or CRYAB exosome in the manufacture of a medicament or a kit for preventing, alleviating or treating a paroxysmal brain disease, wherein the amino acid sequence of the CRYAB is shown as SEQ ID NO: 1 and the nucleotide sequence of the CRYAB is shown as SEQ ID NO:

2.

2. The use according to claim 1, wherein the CRYAB derivative comprises a conjugate of CRYAB and / or a pharmaceutically acceptable salt of CRYAB.

3. The use according to claim 1, wherein the paroxysmal brain disease refers to a brain disease with paroxysmal characteristics, which is manifested by seizures and / or non-seizures.

4. The use according to claim 1, wherein the paroxysmal brain disease is epilepsy.

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