Application of inflammasome NLRP6 in epilepsy treatment

Abstract

The invention discloses an application of inflammasome NLRP6 as a target spot in screening a medicine for treating epilepsy and an application of an NLRP6 expression inhibitor in preparing the medicine for treating epilepsy. The invention discloses the effect of the inflammasome NLRP6 in the aspect of epilepsy neuroinflammation for the first time. The invention researches and determines that the NLRP6 is used as a key regulatory factor of neuroinflammation in epilepsy, and researches the effect of the NLRP6 in activating a caspase-1 / IL-1beta / IL-18 signal channel. The NLRP6 is knocked down, so that the damage effect of epilepsy on neuronal cells can be improved, and the epileptic seizure is improved; overexpression of NLRP6 may aggravate epileptic seizure, neuron injury and neuroinflammatory response. The invention provides a new potential target for epilepsy treatment, and provides a new research thought and direction.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of inflammasome NLRP6 in the treatment of epilepsy. Background Art

[0002] Epilepsy is a common neurological disease characterized by highly synchronous abnormal discharges of neurons in the brain. It is also one of the major diseases that seriously endanger human health. Currently, one-third of epilepsy patients are still resistant to anti-epileptic seizure drugs (ASMs), and ASMs can only relieve symptoms but cannot inhibit epilepsy. More effective drugs are still needed for epilepsy treatment.

[0003] Inflammasomes are a protein complex produced in bone marrow innate immune cells and are an important component of the natural immune system. It mainly consists of three parts: receptor protein, adaptor protein ASC (apoptosis-associated microparticle protein) and downstream Caspase family. Receptor proteins are divided into NOD-like receptor (NLR) family and PYHIN family, including NLRP1, NLRP2, NLRP3, NLRP6, NLRC4 and NLRP12. What they have in common is that they all contain nucleotide binding domain (NBD) and leucine-rich repeat sequence (LRR). Some NLRs also contain pyrin domain (PYD) or caspase activation and recruitment domain (CARD).

[0004] Inflammasomes can recruit pro-caspase-1, regulate the activity (cleavage) of caspase-1 and activate caspase-1. After caspase-1 is activated, it will cut the inactive pro-inflammatory cytokines pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18. In addition, activated Caspase-1 can also cleave GasderminD, allowing it to insert into the membrane, form pores and induce cell pyroptosis.

[0005] As a member of the nucleotide-binding oligomerization domain-like receptor (NLR) family, the NLRP6 inflammasome is a cytoplasmic intrinsic immune sensor responsible for detecting molecular patterns associated with microorganisms. Upon activation, NLRP6 is able to recruit the adaptor protein apoptosis-associated speck-like protein (ASC) and the inflammatory factors caspase-1 or caspase-11. The inflammasome is then formed, promoting the maturation and secretion of proinflammatory cytokines such as IL-18 and IL-1β. Precise regulation of NLRP6 is essential for maintaining tissue homeostasis, as dysregulated activation of inflammasomes may lead to the development of a variety of diseases. NLRP6 inflammasomes are also present in the central and peripheral nervous systems. The assembly and subsequent activation of caspase-1 mediated by NLRP6 promotes disease progression. At present, there are few reports on the role of NLRP6 in neurological diseases. Summary of the invention

[0006] The purpose of the present invention is to address the above-mentioned problems and provide an application of inflammasome NLRP6 in the treatment of epilepsy.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

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

[0009] The drug inhibits the expression of NLRP6.

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

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

[0012] (2) reduce the expression level of pro-inflammatory cytokines;

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

[0014] (4) Improve neuroinflammation;

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

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

[0017] A second aspect of the present invention provides use of an NLRP6 expression inhibitor in the preparation of a drug 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:

[0020] Sense strand: 5'-GGAGCTCGCTTGCTAGTGActcgag-3',

[0021] Antisense strand: 5′-TCACTAGCAAGCGAGCTCCttttt-3′.

[0022] The drug reduces the expression of NLRP6 and hinders the recruitment and assembly of NLRP6 inflammasome by blocking the NLRP6 recruitment of caspase-1 / ASC pathway.

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

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

[0025] (2) reduce the expression level of pro-inflammatory cytokines;

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

[0027] (4) Improve neuroinflammation;

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

[0029] The beneficial effects of the present invention are:

[0030] The present invention reveals for the first time the role of inflammasome NLRP6 in epilepsy neuroinflammation, which may hinder the recruitment and assembly of NLRP6 inflammasome by blocking the caspase-1 / ASC pathway recruited by NLRP6. The present invention study identified NLRP6 as a key regulator of neuroinflammation in epilepsy and studied its role in activating the caspase-1 / IL-1β / IL-18 signaling pathway. Knocking down NLRP6 can improve the damaging effects of epilepsy on neuronal cells, thereby improving epileptic seizures; overexpression of NLRP6 may aggravate epileptic seizures, neuronal damage and neuroinflammatory responses. The present invention provides new potential targets for the treatment of epilepsy and provides new research ideas and directions. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2Figure 5: NLRP6 regulates seizure activity and affects neuronal death after SE. (A) Experimental timeline.

[0033] (B) Whole-brain GFP-tagged AAV expression at week 4, scale bar = 50 μm; (C, E) Weekly spontaneous seizure (SRS) counts; (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 Shown are: 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 between 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 shows pro-inflammatory cytokines in epileptic mice: 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 DESCRIPTION

[0037] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0039] Example 1

[0040] 1 Materials and Methods

[0041] 1.1 Experimental animals

[0042] Male C57BL / 6 mice, 8 weeks old and weighing approximately 25–30 g, were obtained from the Experimental Animal Center of Chongqing Medical University and housed in a standardized SPF environment. 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) was used for Western blot, rabbit anti-NLRP6 antibody (1:1000; Introvigen, USA) was used for immunofluorescence staining, 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), rabbit anti-GAPDH antibody (1:5000; Proteintech, China). In addition, other antibodies used included neuronal nuclear antigen (NeuN, Proteintech, China), glial fibrillary acidic protein (GFAP, Proteintech, China), and ionized calcium binding adaptor protein 1 (Iba1, Servicebio, China). The antibodies used in this study were used according to the instructions, and the molecular weights observed in Western blot were consistent with the literature.

[0045] 1.3 AAV construction and virus injection

[0046] The 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), 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.) contained a specific neuronal promoter and carried a siRNA sequence for knocking down NLRP6 or a NLRP6 overexpression cassette.

[0048] The nucleotide sequence of NLRP6 siRNA is:

[0049] Sense strand (SEQ ID NO. 1): 5'-GGAGCTCGCTTGCTAGTGActcgag-3',

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

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

[0052] Mice were anesthetized with sodium pentobarbital (50 mg / kg) and fixed on a stereotaxic apparatus (Shenzhen Revi Life Sciences Co., Ltd., China). 0.5 μl of AAV-NLRP6-RNAi or AAV-NC-RNAi was injected into the dentate gyrus (DG) and angular gyrus 1 (CA1) regions of the right hippocampus using a 5 μl syringe (Hamilton, Reno, NV, USA) at an injection rate of 0.05 μl / min. The injection coordinates were: point 1 [anteroposterior (AP), -2.0 mm; lateral (ML), -1.5 mm; superior and inferior (DV), -2.0 mm]; point 2 [AP: -2.0 mm; ML: -1.5 mm; DV: -1.5 mm]. The syringe was maintained for 10 minutes after the injection was completed to reduce backflow of the injection track. Subsequently, the same operation was performed on the left hippocampus.

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

[0054] KA model was established 3 weeks after AAV injection. Mice were anesthetized with sodium pentobarbital (50 mg / kg) and fixed on a stereotaxic instrument. 1.0 nmol kainate (KA) (Sigma-Aldrich, USA) was dissolved in 50 nl of 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. After injection, the syringe remained in place for 5 minutes to reduce drug reflux. 2 hours after KA injection, diazepam was used to terminate nonconvulsive epilepsy (SE). Video monitoring recorded the epileptic seizures of mice within 1 month, and spontaneous and recurrent epileptic seizures (SRS) were evaluated according to the Racine score. Next, the head of the experimental mouse was fixed on a stereotaxic instrument, the anterior bregma was exposed, and two small holes were drilled in the midline of the orbit in front of the coronal suture of the skull, and 1 mm diameter screws were implanted to connect the ground wire and fix the electrode. Then a small iron plate was fixed on the side where KA was injected, and the hippocampal area was exposed. After the mice woke up, the brain iron plate was fixed on the local field potential detector, and the microelectrode was slowly inserted into the hippocampus [AP: -2.0mm; ML: -1.5mm; DV: -1.5mm] and connected to the LFP recording system and reference electrode. After turning on the signal amplifier, the local field potential (LFP) signal of the hippocampus of the KA-injected mice was recorded, and the electrical signal and the corresponding spectrum were analyzed using NeuroExplorer software.

[0055] 1.5 Tissue Collection

[0056] After 30 days of TLE (temporal epilepsy), mice were anesthetized with an intraperitoneal injection of 0.8% sodium pentobarbital and rapidly decapitated. Hippocampal tissues were removed and stored at -80°C until further analysis. For transmission electron microscopy (TEM) analysis, six 1 mm 3 The hippocampal tissue samples were taken and imaged at high resolution. Meanwhile, another six samples were fixed with 10% formalin for 12 hours. Then, all formalin-fixed tissue samples were dehydrated and precisely cut into 20 μm thick sections by Leica CM1850 cryostat. Then, the sections were stained according to the standard tissue staining protocol for comprehensive analysis.

[0057] 1.6 Western blot experiment

[0058] Hippocampal tissue was isolated from mice, and protein was extracted using a protein solution containing 5X SDS sampling buffer. The concentration of each protein was 30-50 μg, and 8-12% SDS-PAGE gel was used for electrophoresis separation. The protein was transferred to a PVDF membrane, blocked with 5% skim milk powder for 2 hours at room temperature, and then incubated overnight at 4°C. The next day, the secondary antibody was incubated at room temperature for 2 hours. If necessary, a composite ECL colorimetric reagent (Millipore, WBULP) was used for color development. Subsequently, the optical density of the protein band was quantitatively analyzed using ImageJ software.

[0059] 1.7 Immunofluorescence staining

[0060] Take consecutive coronal sections, block with 5% bovine serum albumin (BSA) at 37°C for 1 hour, then break the cell membrane with 0.4% Triton-X 100 and treat for 60 minutes. Use citric acid repair solution for antigen repair, and then block the sections with ready-made goat serum for 2 hours at 37°C. After preparing the primary antibody, incubate the sections at 37°C overnight. After heating the sections to 37°C for 1 hour, wash them 3 times with 0.01M PBS for 5 minutes each. Then add 20μl of the secondary antibody working solution, incubate at 37°C for 1 hour, and then wash the sections with 0.01M PBS for 10 minutes. Finally, use a DAPI-containing mounting medium to seal the sections and observe and take pictures.

[0061] 1.8 Nissl staining

[0062] The slices were degreased with xylene, stained with Nissl staining solution, stained dark blue, and then rinsed with double distilled water. An appropriate amount of differentiation solution was added for differentiation, and then gradient alcohol dehydration was performed. After degreasing with xylene, the slices were finally sealed with neutral gum. After the slices were dried in a fume hood for 2 hours, they were observed and photographed under a microscope.

[0063] 2. Results

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

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

[0066] 2.2 NLRP6 regulates seizure activity and affects neuronal death after nonconvulsive epilepsy (SE)

[0067] To evaluate whether changes in NLRP6 levels affect seizure activity, AAVs ( Figure 2 A). NLRP6 knockdown (NLRP6-KD) and overexpression (NLRP6-OE) viruses were injected into the CA1 and DG regions of the hippocampus of mice. Autofluorescence of AAV in the hippocampus confirmed the success of viral infection ( Figure 2 B). Four weeks after KA injection, continuous behavioral monitoring showed that NLRP6 knockdown reduced the frequency of SRS ( Figure 2 C) and prolonged the latency period to the first attack ( Figure 2 D). In contrast, NLRP6 overexpression increased the frequency of SRS ( Figure 2 E) and shorten the latency period of epileptic seizures ( Figure 2 F). LFP recording ( 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 increased SLEs ( Figure 2 KN). This suggests a positive correlation between NLRP6 expression and seizure activity in epileptic mice.

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

[0069] Four weeks later, KA-injected mice were subjected to Nissl staining to assess neuronal death. Neuronal loss in the hippocampus was aggravated in TLE mice ( Figure 3 A). NLRP6 knockdown alleviated neuronal death, whereas NLRP6 overexpression aggravated 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 NLRP6 intervention regulates the assembly of NLRP6 inflammasome ASC and caspase-1

[0071] We evaluated the expression levels of structural components of the NLRP6 inflammasome. Western blot analysis showed that the levels of adaptor protein (ASC) and caspase-1p20 / caspase-1 were increased in epileptic mice compared with wild-type (WT) controls. Figure 4 A), while the levels of these proteins were decreased in NLRP6-OE( Figure 4 These results suggest that knockdown of NLRP6 can effectively reverse this increase, while overexpression of NLRP6 aggravates this phenomenon ( Figure 4 CF). Our results indicate that the three core components of the NLRP6 inflammasome are fully assembled in epileptic mice.

[0072] 2.5 NLRP6 / ASC / caspase-1 complex regulates epileptic neuroinflammation after NLRP6 intervention

[0073] Western blot analysis showed that ( Figure 5 ), the levels of proinflammatory cytokines IL-18, IL-1β, and IL-6 were increased in epileptic mice compared with the WT control group. In the NLRP6-KD group, the expression levels of these cytokines were significantly reduced, indicating that NLRP6 knockdown can alleviate neuroinflammation. In contrast, the cytokine expression in the NLRP6-OE group was increased, resulting in aggravated neuroinflammation. Our results suggest that the NLRP6 inflammasome and its core components ASC and caspase-1 play a role in regulating the production of proinflammatory cytokines in TLE.

[0074] 3 Analysis

[0075] As a member of the NLR (NOD-like receptor) family, NLRP6 plays a vital role in intestinal, cancer and neurological diseases. Although the function of NLRP6 inflammasome in the nervous system has been reviewed, its specific regulatory role in epilepsy has not been deeply explored. This study provides new insights into the role of NLRP6 in the pathogenesis of epilepsy, especially its involvement in neuroinflammation.

[0076] At the same time, studies have found that NLRP6 expression is upregulated in human brain slices from patients with cerebral hemorrhage. Our results also showed that in KA-induced epileptic mice, NLRP6 expression was significantly increased in the cerebral cortex and hippocampus, which provides important clues for understanding the molecular mechanisms of epilepsy susceptibility. A recent in vitro study showed that under ethanol exposure, the aggregation of NLRP6 inflammasomes in astrocytes induced the production of proinflammatory cytokines IL-1β and IL-18. The main localization of NLRP6 in hippocampal neurons suggests that it may directly affect neuronal excitability and inflammatory responses in the focal area of ​​epilepsy.

[0077] This study identified 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 epileptic seizures, neuronal damage, and neuroinflammatory responses. These results suggest that NLRP6 may be a promising therapeutic target. Future studies could focus on elucidating the specific cellular pathways regulated by NLRP6 and evaluating the clinical efficacy of therapeutic approaches targeting the inflammasome.

Claims

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

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

3. The use according to claim 2, characterized in that: The drug has any of the following functions: (1) Reduce damage to neuronal cells and alleviate neuronal death; (2) reduce the expression level of pro-inflammatory cytokines; (3) hindering the recruitment and assembly of ASC and caspase-1, the core components of the NLRP6 inflammasome; (4) Improve neuroinflammation; (5) Reduce the frequency of recurrent epileptic seizures.

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

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

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

7. The use according to claim 6, characterized in that: The nucleotide molecule includes siRNA, and the nucleotide sequence of the siRNA is: Sense strand: 5'-GGAGCTCGCTTGCTAGTGActcgag-3', Antisense strand: 5′-TCACTAGCAAGCGAGCTCCttttt-3′.

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

9. The use according to claim 5, characterized in that: The expression inhibitor has any of the following functions: (1) Reduce damage to neuronal cells and alleviate neuronal death; (2) reduce the expression level of pro-inflammatory cytokines; (3) hindering the recruitment and assembly of ASC and caspase-1, the core components of the NLRP6 inflammasome; (4) Improve neuroinflammation; (5) Reduce the frequency of recurrent epileptic seizures.

Images