Application of inflammasome NLRP6 in the treatment of epilepsy

Abstract

This invention discloses the application of the inflammasome NLRP6 as a target in screening drugs for treating epilepsy, and the application of NLRP6 expression inhibitors in the preparation of drugs for treating epilepsy. This invention reveals for the first time the role of the inflammasome NLRP6 in epileptic neuroinflammation. This invention identifies NLRP6 as a key regulator of neuroinflammation in epilepsy and investigates its role in activating the caspase-1 / IL-1β / IL-18 signaling pathway. Knockdown of NLRP6 can improve the damaging effects of epilepsy on neurons, thereby improving seizures; overexpression of NLRP6 may exacerbate seizures, neuronal damage, and neuroinflammatory responses. This invention provides a new potential target for epilepsy treatment, offering new research ideas and directions.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of the inflammasome NLRP6 in the treatment of epilepsy. Background Technology

[0002] Epilepsy is a common neurological disorder characterized by highly synchronized abnormal electrical discharges in the brain, and it is one of the major diseases that seriously endanger human health. Currently, one-third of epilepsy patients are resistant to anti-epileptic drugs (ASMs), which can only relieve symptoms and cannot suppress seizures. The development of more effective drugs is still needed for epilepsy treatment.

[0003] Inflammasomes are protein complexes produced in innate immune cells of the bone marrow and are an important component of the innate immune system. They mainly consist of three parts: receptor proteins, adaptor proteins ASC (apoptosis-associated microparticle proteins), and downstream caspase families. Receptor proteins are divided into the NOD-like receptor (NLR) family and the PYHIN family, including NLRP1, NLRP2, NLRP3, NLRP6, NLRC4, and NLRP12. They all share the characteristic of containing a nucleotide-binding domain (NBD) and leucine-rich repeat sequences (LRR). Some NLRs also contain a heat protein domain (PYD) or a caspase activation and recruitment domain (CARD).

[0004] Inflammasomes can recruit pro-caspase-1, regulate its activity (cleavage), and activate it. Once activated, caspase-1 cleaves inactive pro-inflammatory cytokines pro-IL-1β and pro-IL-18 into mature forms IL-1β and IL-18. Furthermore, activated caspase-1 can cleave Gasdermin D, causing it to insert into the membrane, forming pores and inducing pyroptosis.

[0005] As a member of the nucleotide-binding oligomerization domain-like receptor (NLR) family, the NLRP6 inflammasome is an intrinsic cytoplasmic immune sensor responsible for detecting microbe-associated molecular patterns. Upon activation, NLRP6 recruits the adaptor protein apoptosis-associated speckle-like protein (ASC) and the inflammatory cytokines caspase-1 or caspase-11. This subsequently leads to the formation of the inflammasome, promoting the maturation and secretion of pro-inflammatory cytokines such as cytokine-18 (IL-18) and cytokine-1β (IL-1β). Precise regulation of NLRP6 is crucial for maintaining tissue homeostasis, as dysregulated activation of the inflammasome can contribute to the development of various diseases. The NLRP6 inflammasome is also present in the central and peripheral nervous systems. NLRP6-mediated assembly and subsequent activation of caspase-1 promotes disease progression. Currently, there are limited reports on the role of NLRP6 in neurological diseases. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned problems by providing an application of the inflammasome NLRP6 in the treatment of epilepsy.

[0007] To achieve its objective, the present invention employs the following technical solution:

[0008] A first aspect of the present invention provides the use of the inflammasome NLRP6 as a target in screening drugs for the treatment of epilepsy.

[0009] The drug inhibits the expression of NLRP6.

[0010] The drug has any of the following functions:

[0011] (1) Reduce damage to neurons and alleviate neuronal death;

[0012] (2) Reduce the expression levels of pro-inflammatory cytokines;

[0013] (3) It inhibits the recruitment and assembly of ASC and caspase-1, the core components of the NLRP6 inflammasome;

[0014] (4) Improves nerve inflammation;

[0015] (5) Reduce the frequency of recurrent epileptic seizures.

[0016] The pro-inflammatory cytokines include IL-18, IL-1β, and IL-6.

[0017] A second aspect of the invention provides the use of NLRP6 expression inhibitors in the preparation of medicaments for treating epilepsy.

[0018] The expression inhibitors include compounds, proteins, and nucleotide molecules.

[0019] Preferably, the nucleotide molecule comprises siRNA, and the nucleotide sequence of the siRNA is as follows:

[0020] Justice Chain: 5'-GGAGCTCGCTTGCTAGTGActcgag-3'

[0021] Antonym chain: 5'-TCACTAGCAAGCGAGCTCCttttt-3'.

[0022] The drug reduces NLRP6 expression, thereby blocking the recruitment and assembly of NLRP6 inflammasomes by inhibiting the NLRP6 recruitment caspase-1 / ASC pathway.

[0023] The expression inhibitor has any of the following functions:

[0024] (1) Reduce damage to neurons and alleviate neuronal death;

[0025] (2) Reduce the expression levels of pro-inflammatory cytokines;

[0026] (3) It inhibits the recruitment and assembly of ASC and caspase-1, the core components of the NLRP6 inflammasome;

[0027] (4) Improves nerve inflammation;

[0028] (5) Reduce the frequency of recurrent epileptic seizures.

[0029] The beneficial effects of this invention are:

[0030] This invention reveals for the first time the role of the inflammasome NLRP6 in epileptic neuroinflammation, potentially by blocking the NLRP6 recruitment of the caspase-1 / ASC pathway, thereby hindering NLRP6 inflammasome recruitment and assembly. This invention identifies NLRP6 as a key regulator of neuroinflammation in epilepsy and investigates its role in activating the caspase-1 / IL-1β / IL-18 signaling pathway. Knockdown of NLRP6 can ameliorate the damaging effects of epilepsy on neurons, thereby improving seizure frequency; overexpression of NLRP6 may exacerbate seizures, neuronal damage, and neuroinflammatory responses. This invention provides a novel potential target for epilepsy treatment, offering new research ideas and directions. Attached Figure Description

[0031] Figure 1 The expression and localization of NLRP6 in the cerebral cortex (A) and hippocampus (B) of epileptic mice are shown (C).

[0032] Figure 2This demonstrates that NLRP6 modulates seizure activity and influences neuronal death after seizures: (A) Experimental timeline;

[0033] (B) Whole-brain GFP-labeled AAV expression at week 4, scale bar = 50 μm; (C, E) Weekly spontaneous seizure (SRS) count; (D, F) Seizure latency; (G, K) Representative local field potential (LFP) waveforms; (HJ, LN) Frequency and duration of pathological discharge events (SLEs) in each group (n = 5).

[0034] Figure 3 The results show: A. Nissl staining results; B. Quantitative analysis of the number of Nissl+ cells in the hippocampus; *P<0.05, **P<0.01, ***P<0.001, compared with the control group; #P<0.05, comparison of con-KD and NLRP6-KD.

[0035] Figure 4 Western blot analysis of ASC and caspase-1p20 / caspase-1 levels in epileptic mice: A.-B. Representative Western blot images; CF. Western blot analysis of protein levels.

[0036] Figure 5 Western blot analysis of pro-inflammatory cytokines in mice with epilepsy: A.-B. Representative Western blot images; CH. Western blot analysis of the expression levels of pro-inflammatory cytokines IL-18, IL-1β and IL-6. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0038] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0039] Example 1

[0040] 1. Materials and Methods

[0041] 1.1 Laboratory Animals

[0042] Eight-week-old male C57BL / 6 mice, weighing approximately 25-30g, were used and housed in a standardized SPF (Special Protected Forest) environment at the Experimental Animal Center of Chongqing Medical University. All experimental procedures were approved by the Animal Experiment Ethics Committee of Chongqing Medical University and followed international standards.

[0043] 1.2 Experimental Reagents

[0044] Rabbit anti-NLRP6 antibody (1:1000; Abclonal, China) for Western blot, rabbit anti-NLRP6 antibody for immunofluorescence staining (1:1000; Introvigen, USA), rabbit anti-ASC antibody (1:1000; Proteintech, China), rabbit anti-Caspase-1 antibody (1:1000; MCE, USA), rabbit anti-IL-1β antibody (1:1000; Proteintech, China), rabbit anti-IL-18 antibody (1:1000; Proteintech, China), mouse anti-IL-6 antibody (1:1000; Santa, USA), and rabbit anti-GAPDH antibody (1:5000; Proteintech, China). Other antibodies used include neuronal nuclear antigen (NeuN, Proteintech, China), glial fibrillary acidic protein (GFAP, Proteintech, China), and ionized calcium-binding aptamer protein 1 (Iba1, Servicebio, China). All antibodies used in this study were used according to their instructions, and the molecular weights observed in Western blots were consistent with those in the literature.

[0045] 1.3 AAV Construction and Virus Injection

[0046] Mice were randomly divided into 5 groups: untreated control group (Control group), Con-AAV-KD and KA treatment group (Con-KD group, i.e., empty vector AAV injection + KA modeling group), NLRP6-AAV-KD and KA treatment group (NLRP6-KD group, i.e., NLRP6 knockdown AAV injection + KA modeling group), Con-AAV-OE and KA treatment group (Con-OE group, i.e., empty vector AAV injection + KA modeling group), and NLRP6-AAV-OE and KA treatment group (NLRP6-OE group, i.e., NLRP6 overexpression AAV injection + KA modeling group).

[0047] The ordered AAV (Beijing Qingke Biotechnology Co., Ltd.) contains a specific neuronal promoter and carries an siRNA sequence that knocks down NLRP6 or an NLRP6 overexpression cassette.

[0048] The nucleotide sequence of NLRP6 siRNA is as follows:

[0049] Chain of Justice (SEQ ID NO.1): 5'-GGAGCTCGCTTGCTAGTGActcgag-3'

[0050] Antisense chain (SEQ ID NO.2): 5'-TCACTAGCAAGCGAGCTCCttttt-3'.

[0051] The nucleotide sequence of NLRP6 overexpression is shown in SEQ ID NO.3.

[0052] Mice were anesthetized with sodium pentobarbital (50 mg / kg) and fixed on a stereotactic apparatus (Shenzhen Ruiwei Life Science Co., Ltd., China). Using a 5 μl syringe (Hamilton, Reynolds, Nevada, USA), 0.5 μl of AAV-NLRP6-RNAi or AAV-NC-RNAi was injected into the right hippocampal dentate gyrus (DG) and angular gyrus 1 (CA1) regions, respectively, at an injection rate of 0.05 μl / min. Injection coordinates were: Point 1 [AP, -2.0 mm; ML, -1.5 mm; DV, -2.0 mm]; Point 2 [AP: -2.0 mm; ML: -1.5 mm; DV: -1.5 mm]. The syringe was held for 10 minutes after injection to reduce reflux along the injection path. The same procedure was then performed on the left hippocampus.

[0053] 1.4KA-induced mouse seizure model, behavioral video monitoring, and local field potential (LFP) recording

[0054] Three weeks after AAV injection, a kaempferol (KA) model was established in mice. Mice were anesthetized with sodium pentobarbital (50 mg / kg) and fixed to a stereotactic apparatus. 1.0 nmol kaempferol (KA) (Sigma-Aldrich, USA) was dissolved in 50 nmol saline using a 0.5 μl syringe and injected into the right hippocampus [AP: -2.0 mm; ML: -1.5 mm; DV: -1.5 mm] for 3 minutes. The syringe was held in place for 5 minutes after injection to reduce drug reflux. Two hours after KA injection, diazepam was used to terminate nonconvulsive continuous seizures (SE). Video monitoring recorded seizure activity over one month, and spontaneous and recurrent seizures (SRS) were assessed using the Racine score. Next, the mouse head was fixed to the stereotactic apparatus, exposing the anterior fontanelle. Two small holes were drilled at the midline of the orbit anterior to the coronal suture of the skull, and 1 mm diameter screws were inserted to connect the ground wire and fix the electrodes. A small iron plate was then fixed to the side where KA was injected, exposing the hippocampal region. After the mice regained consciousness, the brain plate was fixed to the local field potential detector, and the microelectrode was slowly inserted into the hippocampus [AP: -2.0 mm; ML: -1.5 mm; DV: -1.5 mm] and connected to the LFP recording system and reference electrode. After the signal amplifier was turned on, the local field potential (LFP) signal of the hippocampus of the mouse on the KA side was recorded, and the electrical signal and corresponding spectrogram were analyzed using NeuroExplorer software.

[0055] 1.5 Organization and Collection

[0056] Thirty days after TLE (time-dependent epilepsy), mice were anesthetized with an intraperitoneal injection of 0.8% sodium pentobarbital and rapidly decapitated. Hippocampal tissue was removed and stored at -80°C until further analysis. For transmission electron microscopy (TEM) analysis, six 1mm... 3 Hippocampal tissue samples were collected and imaged at high resolution. Simultaneously, six other samples were fixed in 10% formalin for 12 hours. All formalin-fixed tissue samples were then dehydrated using a Leica CM1850 cryostat and precisely sectioned to a thickness of 20 μm. The sections were then stained according to standard tissue staining protocols for comprehensive analysis.

[0057] 1.6 Western blot experiment

[0058] Hippocampal tissue was isolated from mice, and proteins were extracted using a protein solution containing 5X SDS sampling buffer at a concentration of 30-50 μg per group. Separation was performed by electrophoresis on an 8-12% SDS-PAGE gel. Proteins were transferred to PVDF membranes, blocked with 5% skim milk at room temperature for 2 hours, and then incubated overnight at 4°C. The next day, the membranes were incubated with a secondary antibody at room temperature for 2 hours. If necessary, color development was performed using a multi-component ECL chromogenic reagent (Millipore, WBULP). Subsequently, the optical density of the protein bands was quantitatively analyzed using ImageJ software.

[0059] 1.7 Immunofluorescence staining

[0060] Serial coronal sections were blocked with 5% bovine serum albumin (BSA) at 37°C for 1 hour, followed by cell membrane disruption with 0.4% Triton-X 100 for 60 minutes. Antigen retrieval was performed using citrate retrieval buffer, and the sections were then blocked with ready-made goat serum at 37°C for 2 hours. After preparing the primary antibody, the sections were incubated overnight at 37°C. After heating the sections to 37°C for 1 hour, they were washed three times with 0.01M PBS for 5 minutes each time. Then, 20 μl of secondary antibody working solution was added, and the sections were incubated at 37°C for 1 hour, followed by washing with 0.01M PBS for 10 minutes. Finally, the sections were mounted using a DAPI-containing mounting medium, observed, and photographed.

[0061] 1.8Nissl staining

[0062] Sections were defatted with xylene, stained with Nissl staining solution until deep blue, and then rinsed with double-distilled water. Differentiation was initiated with an appropriate amount of differentiation solution, followed by graded alcohol dehydration, defatting with xylene, and finally mounting with neutral resin. After drying in a fume hood for 2 hours, the sections were observed and photographed under a microscope.

[0063] 2. Results

[0064] 2.1 Expression and localization of NLRP6 in a mouse model of epilepsy

[0065] First, a chronic epilepsy mouse model was established. Compared with the control group, NLRP6 expression in the hippocampus and cerebral cortex of the epileptic mice was significantly upregulated. Figure 1 AB, P < 0.05). NLRP6 mainly co-localizes with the neuronal marker NeuN, and is also detected in small amounts in Iba1-positive microglia. Figure 1 C).

[0066] 2.2 NLRP6 modulates seizure activity and influences neuronal death after nonconvulsive sustained epilepsy (SE).

[0067] To assess whether changes in NLRP6 levels affect seizure activity, AAVs were administered three weeks prior to KA-induced chronic seizures. Figure 2 A). NLRP6 knockdown (NLRP6-KD) and overexpression (NLRP6-OE) viruses were injected into the CA1 and DG regions of the hippocampus in mice. Autofluorescence of AAV in the hippocampus confirmed successful viral infection. Figure 2 B). Four weeks after KA injection, ongoing behavioral monitoring showed that NLRP6 knockdown reduced the frequency of SRS ( Figure 2 C), and prolonged the latency period of the first attack ( Figure 2 D). Conversely, NLRP6 overexpression increases the frequency of SRS ( Figure 2 E) and shortened the latency period of epileptic seizures ( Figure 2 F). LFP record ( Figure 2 GJ) showed that, compared with the control group, the NLRP6-KD group had fewer seizure-like events (SLEs), while the NLRP6-OE group had an increase in SLEs. Figure 2 This indicates a positive correlation between NLRP6 expression and seizure activity in epileptic mice.

[0068] 2.3 NLRP6 knockdown reduces susceptibility to KA-induced seizures and alleviates neuronal apoptosis.

[0069] Four weeks later, Nissl staining was performed on mice injected with KA to assess neuronal death. In TLE mice, neuronal loss in the hippocampus was aggravated. Figure 3 A). NLRP6 knockdown alleviated neuronal death, while NLRP6 overexpression exacerbated neuronal death. Figure 3 B). Overall, these results indicate that NLRP6 knockdown reduces susceptibility to KA-induced seizures and alleviates neuronal apoptosis.

[0070] 2.4 Regulation of NLRP6 inflammasome ASC and caspase-1 assembly after NLRP6 intervention

[0071] We assessed the expression levels of structural components of the NLRP6 inflammasome. Western blot analysis showed increased levels of adaptor protein (ASC) and caspase-1 p20 / caspase-1 in epileptic mice compared to wild-type (WT) controls. In the NLRP6-KD group ( Figure 4 In A), the levels of these proteins decreased, while in NLRP6-OE ( Figure 4 In group B), its expression level was elevated. These results indicate that NLRP6 knockdown can effectively reverse this elevation, while NLRP6 overexpression exacerbates this phenomenon. Figure 4 Our results indicate that the three core components of the NLRP6 inflammasome are fully assembled in epileptic mice.

[0072] 2.5 NLRP6 intervention followed by NLRP6 intervention modulates epileptic neuroinflammation.

[0073] Western blot analysis showed that ( Figure 5 Compared with the WT control group, the levels of pro-inflammatory cytokines IL-18, IL-1β, and IL-6 were increased in epileptic mice. In the NLRP6-KD group, the expression levels of these cytokines were significantly reduced, indicating that NLRP6 knockdown alleviates neuroinflammation. Conversely, the NLRP6-OE group showed increased cytokine expression, leading to exacerbation of neuroinflammation. Our results suggest that the NLRP6 inflammasome and its core components ASC and caspase-1 play a role in regulating the production of pro-inflammatory cytokines in transient ischemic attacks (TLE).

[0074] 3 Analysis

[0075] As a member of the NLR (NOD-like receptor) family, NLRP6 plays a crucial role in intestinal, cancer, and neurological diseases. While the function of the NLRP6 inflammasome in the nervous system has been reviewed, its specific regulatory role in epilepsy remains largely unexplored. This study provides new insights into the role of NLRP6 in the pathogenesis of epilepsy, particularly its involvement in neuroinflammation.

[0076] Meanwhile, studies have found that NLRP6 expression is upregulated in human brain slices from patients with cerebral hemorrhage. Our results similarly indicate that NLRP6 expression is significantly increased in the cerebral cortex and hippocampus of KA-induced epileptic mice, providing important clues for understanding the molecular mechanisms of epilepsy susceptibility. A recent in vitro study showed that NLRP6 inflammasome accumulation in astrocytes under ethanol exposure induces the production of pro-inflammatory cytokines IL-1β and IL-18. The major localization of NLRP6 in hippocampal neurons suggests that it may directly affect neuronal excitability and inflammatory responses in epileptic focal regions.

[0077] This invention identifies NLRP6 as a key regulator of neuroinflammation in epilepsy, highlighting its role in activating the caspase-1 / IL-1β / IL-18 signaling pathway. Overexpression of NLRP6 may exacerbate seizures, neuronal damage, and neuroinflammatory responses. These results suggest that NLRP6 may be a promising therapeutic target. Future research could focus on elucidating the specific cellular pathways regulated by NLRP6 and evaluating the clinical efficacy of therapies targeting the inflammasome.

Claims

1. Application of inflammasome NLRP6 as a target in screening drugs for the treatment of epilepsy.

2. The application according to claim 1, characterized in that: The drug inhibits the expression of NLRP6.

3. The application according to claim 2, characterized in that: The drug has any of the following functions: (1) Reduce damage to neurons and alleviate neuronal death; (2) Reduce the expression levels of pro-inflammatory cytokines; (3) It inhibits the recruitment and assembly of ASC and caspase-1, the core components of the NLRP6 inflammasome; (4) Improves nerve inflammation; (5) Reduce the frequency of recurrent epileptic seizures.

4. The application according to claim 3, characterized in that: The pro-inflammatory cytokines include IL-18, IL-1β, and IL-6.

5. Application of NLRP6 expression inhibitors in the preparation of drugs for treating epilepsy.

6. The application according to claim 5, characterized in that: The expression inhibitors include compounds, proteins, or nucleotide molecules.

7. The application according to claim 6, characterized in that: The nucleotide molecule includes siRNA, and the nucleotide sequence of the siRNA is as follows: Justice Chain: 5'-GGAGCTCGCTTGCTAGTGActcgag-3' Antonym chain: 5'-TCACTAGCAAGCGAGCTCCttttt-3'.

8. The application according to claim 5, characterized in that: The drug reduces NLRP6 expression, thereby blocking the recruitment and assembly of NLRP6 inflammasomes by inhibiting the NLRP6 recruitment caspase-1 / ASC pathway.

9. The application according to claim 5, characterized in that: The expression inhibitor has any of the following functions: (1) Reduce damage to neurons and alleviate neuronal death; (2) Reduce the expression levels of pro-inflammatory cytokines; (3) It inhibits the recruitment and assembly of ASC and caspase-1, the core components of the NLRP6 inflammasome; (4) Improves nerve inflammation; (5) Reduce the frequency of recurrent epileptic seizures.

Images