Application of lncMCL1 in regulating pyroptosis degree of neuronal cells and relieving and treating related diseases

Abstract

The invention provides application of lncMCL1 in regulating and controlling the pyroptosis degree of neuronal cells and relieving and treating related diseases, and relates to the technical field of biological medicine. The invention specifically discloses regulation and control of pyroptosis of neuronal cells by enhancing or reducing expression of lncMCL1, and further discloses application of a substance for regulating and controlling expression enhancement of lncMCL1 in preparation of drugs for relieving and treating neuronal function deficiency related diseases, especially epilepsy. In-vivo experiments and in-vitro experiments prove that lncMCL1 can be used as a key regulatory factor for pyroptosis of neuronal cells by regulating inflammatory response of the neuronal cells, and can effectively regulate pyroptosis of the neuronal cells and relieve and treat diseases related to neuronal function deficiency.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of lncMCL1 in regulating the degree of neuronal pyroptosis and the remission and treatment of related diseases. Background Art

[0002] Epilepsy is a common neurological disorder that affects people of all ages, with a prevalence of approximately 0.7%. Although antiepileptic drugs remain the main treatment option, about one-third of patients cannot adequately control seizures and ultimately develop drug-resistant epilepsy. Temporal lobe epilepsy (TLE) is one of the most common refractory epilepsies, characterized by recurrent and unpredictable seizures. TLE is usually associated with various pathological changes in the hippocampus, including extensive neuronal loss, neural circuit reorganization, and gliosis.

[0003] Programmed cell death (PCD) is a process of self-destruction of cells, which can be triggered by cellular stress and inflammatory responses. PCD includes various forms of cell death, such as apoptosis, autophagy, and programmed necrosis, and the latter is further divided into necroptosis, ferroptosis, and pyroptosis. Pyroptosis is a well-known form of pro-inflammatory programmed cell death and is associated with epilepsy in both animal models and epilepsy patients. Pyroptosis is a type of PCD triggered by the activation of immunocytokines, characterized by a strong pro-inflammatory response. It occurs through two main pathways: the classical (caspase-1-dependent) pathway, in which NLRP3 inflammasome activation leads to caspase-1 activation, cytokine release (such as IL-1β, IL-18), and cell membrane rupture; and the non-classical (caspase-1-independent) pathway, which involves caspases such as Caspase-11, Caspase-4, or Caspase-5. Clinical and animal studies have confirmed increased expression of NLRP3, caspase-1, and IL-1β in the hippocampal tissue of patients with TLE. Notably, pyroptosis mainly occurs in hippocampal neurons and is usually triggered by the abnormal activation of the NLRP3 inflammasome. Recent studies have shown that knocking out NLRP3 can inhibit epilepsy-induced neuronal loss in the CA3 region and the expression of IL-1β, suggesting that the classical pyroptosis pathway dependent on the NLRP3 inflammasome plays an important role in epileptic neuronal loss. Studies have shown that factors such as oxidative stress and transient receptor potential channels regulate the activation of NLRP3 and pyroptosis in epilepsy. In addition, the ubiquitin-proteasome pathway and inflammatory-related pathway initiators such as intestine-enriched Kruppel-like factor and Signal Transducer and Activator of Transcription 3 (STAT3) may also affect the function of NLRP3.

[0004] A large amount of evidence indicates that lncRNA is a key regulator of the NLRP3 inflammasome and pyroptosis. The regulation mediated by lncRNA is related to its subcellular localization. Cytoplasmic lncRNA plays a role in neuronal loss and inflammatory responses in epilepsy. The regulation process includes the activation of the NLRP3 inflammasome, neuronal apoptosis, and the over-activation of astrocytes and microglia. Therefore, seeking effective lncRNA targets that can regulate neuronal pyroptosis and treat epilepsy is one of the technical problems that need to be urgently solved by those skilled in the art. Summary of the Invention

[0005] The present invention provides the application of lncMCL1 in regulating the degree of neuronal pyroptosis and the treatment of epilepsy. lncMCL1 can be used as a new target to regulate the degree of neuronal pyroptosis and the treatment of epilepsy.

[0006] In a first aspect, the present invention provides the application of a substance that enhances the expression of lncMCL1 in neurons in the preparation of a drug for relieving and / or treating diseases, disorders or dysfunctions related to neuronal function deficiency.

[0007] For the application as described above, the substance that enhances the expression of lncMCL1 in neurons is a biological material related to the lncMCL1, and the biological material is selected from at least one of A1)-A4): A1) A nucleic acid molecule encoding the lncMCL1; A2) An expression cassette containing the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2); A4) A recombinant microorganism containing the nucleic acid molecule described in A1), or a recombinant microorganism containing the expression cassette described in A2), or a recombinant microorganism containing the recombinant vector described in A3).

[0008] For the application as described above, the neurons are derived from at least one of the hippocampal tissue and the cerebral cortex.

[0009] For the application as described above, the neurons are derived from humans or non-human primates. Further, the non-human primate can be at least one of rats and mice.

[0010] In a specific embodiment, the neurons are hippocampal neurons.

[0011] In a specific embodiment, when the neurons are derived from the hippocampal tissue of a mouse, the coding sequence of the coding strand of the nucleic acid molecule described in A1) is a cDNA molecule or a DNA molecule having the sequence shown in SEQ ID NO:1.

[0012] For the application as described above, the diseases, disorders or dysfunctions related to neuronal function deficiency are epilepsy. Further, the epilepsy is temporal lobe epilepsy. More specifically, the diseases, disorders or dysfunctions related to neuronal function deficiency can be status epilepticus caused by epilepsy.

[0013] The application as described above enhances the expression of lncMCL1 in the neuronal cells by overexpression. In a specific embodiment, the coding sequence of lncMCL1 can be introduced into an overexpression vector to obtain a recombinant vector containing the lncMCL1 coding sequence, and then the recombinant vector containing the lncMCL1 coding sequence is injected into the hippocampal tissue to enhance the expression of lncMCL1 in the neuronal cells of the hippocampal tissue.

[0014] In a specific embodiment, the overexpression vector can be an adeno-associated virus (AAV) vector or a lentiviral vector that can infect neuronal cells or animal models.

[0015] The application as described above alleviates and / or treats diseases, disorders or dysfunctions related to neuronal function deficiency, which are manifested as at least one of the following: reduced seizure frequency, protection against damage to neuronal cells in the hippocampal tissue, and reduced expression of proteins related to the NLRP3 inflammasome signaling pathway and / or the content of molecular markers of inflammatory necrosis in neuronal cells.

[0016] In a second aspect, the present invention provides a method for regulating the degree of pyroptosis or inflammatory necrosis of neuronal cells, including regulating the expression of lncMCL1 in neuronal cells to regulate the degree of pyroptosis or inflammatory necrosis of the neuronal cells.

[0017] The regulation can be carried out in vivo or in vitro.

[0018] In one embodiment, the regulation is to increase or decrease the degree of pyroptosis or inflammatory necrosis of neuronal cells, depending on the application purpose. Preferably, the regulation is to decrease the degree of pyroptosis or inflammatory necrosis of neuronal cells. The neuronal cells are as described in the first aspect above.

[0019] Inflammatory necrosis and pyroptosis are two forms of cell death. Both are involved in the neuroinflammatory response, can lead to the release of inflammatory factors, activate the inflammatory cascade reaction, and cause neuronal damage and dysfunction. They induce and promote each other, forming a vicious cycle. The inflammatory necrosis of neurons triggers pyroptosis, and the inflammatory factors released by pyroptosis further exacerbate neuroinflammation, leading to more neuronal death and further aggravating the condition of epilepsy. A common feature of the two forms is that they can both activate the inflammasome. For example, it causes an increase in the expression of proteins related to the NLRP3 inflammasome signaling pathway in neuronal cells and / or an increase in the content of molecular markers of inflammatory necrosis. In a specific embodiment, the proteins related to the NLRP3 inflammasome signaling pathway can include at least one of NLRP3, GSDMD-FL, GSDMD-N, pro-Caspase1, c-Caspase1; the molecular markers of inflammatory necrosis can include at least one of TNFα, IL-1β and IL-18.

[0020] In a preferred embodiment, the regulation is achieved by enhancing or reducing the expression of lncMCL1 in neuronal cells. In a specific embodiment, the expression of lncMCL1 in the neuronal cells is enhanced by overexpression. In another specific embodiment, the expression of lncMCL1 in the neuronal cells is reduced by gene editing technology or small interfering RNA technology. Further, the gene editing technology can be CRISPR-Cas9. Further, the double-stranded RNA used in the small interfering RNA technology is selected from at least one of siRNA1 and siRNA2. The siRNA1 includes a first strand with a nucleotide sequence of SEQ ID NO:2 and a second strand with a nucleotide sequence of SEQ ID NO:3. The siRNA2 includes a first strand with a nucleotide sequence of SEQ ID NO:4 and a second strand with a nucleotide sequence of SEQ ID NO:5.

[0021] In one embodiment, the regulation method includes regulating (e.g., reducing) the pyroptosis or inflammatory necrosis degree of neuronal cells by regulating the expression of lncMCL1 in neuronal cells in vitro using the substance for enhancing the expression of lncMCL1 in neuronal cells according to the first aspect.

[0022] In a specific embodiment, compared with before the expression of lncMCL1 is regulated, the expression level of proteins related to the NLRP3 inflammasome signaling pathway and / or the content of molecular markers of inflammatory necrosis in the regulated cells are reduced by more than 30%. Further, they are reduced by 35%, 40%, 45%, 50%, 60% or more. In a specific embodiment, compared with before the expression of lncMCL1 is regulated, the expression level of proteins related to the NLRP3 inflammasome signaling pathway and / or the content of molecular markers of inflammatory necrosis in the regulated cells are increased by more than 30%. Further, they are increased by 35%, 40%, 45%, 50%, 60% or more.

[0023] For the method as described above, the direct purpose of the method is for non-disease diagnosis and / or non-disease treatment purposes. The above application or method is an application or method for non-disease diagnosis. The above application or method does not directly aim to obtain a disease diagnosis result or health condition of a living human body or animal body.

[0024] In a third aspect, the present invention provides a pharmaceutical composition, including a substance for enhancing the expression of lncMCL1 in neuronal cells and a pharmaceutically acceptable carrier.

[0025] For the pharmaceutical composition as described above, the pharmaceutically acceptable carrier can be a substance that helps the said substance effectively enter cells and exert its effects, and can be specifically determined according to factors such as the purpose of treatment, the stability of the substance, the required targeting, and the route of administration.

[0026] In the present invention, according to the drug release mode, the drugs described in the first aspect and the pharmaceutical compositions described in the third aspect can adopt various dosage forms, including, but not limited to, conventional preparations, sustained-release preparations, controlled-release preparations, and various particulate drug delivery systems. In a specific embodiment, the preparation can be in the form of an injection.

[0027] In the fourth aspect, the present invention provides a method for alleviating and / or treating diseases related to neuronal dysfunction, including administering to a subject a therapeutically effective amount of the substance described in the first aspect that enhances the expression of lncMCL1 in neuronal cells.

[0028] For the method as described above, the subject refers to a subject suffering from a disease, disorder, or dysfunction related to neuronal dysfunction, such as a patient clinically diagnosed with epilepsy. Further, the subject suffers from temporal lobe epilepsy. The diseases, disorders, or dysfunctions related to neuronal dysfunction can be status epilepticus caused by epilepsy. In the neurons of the subject with the diseases, disorders, or dysfunctions related to neuronal dysfunction, there may be an inflammatory response, especially the inflammatory response triggered by the classical (caspase-1 dependent) pathway.

[0029] For the method as described above, the subject can be a mammal, and the mammal can be selected from bovidae, equidae, felidae, canidae, leporidae, suidae, camelidae, rodents, and primates, etc., including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, orangutans), and humans, preferably rats, mice, and humans.

[0030] For the method as described above, the dosage of the substance that enhances the expression of lncMCL1 in neuronal cells depends on the mode of administration, the route of administration, the age and / or weight of the individual, and the condition of the treated individual, is variable, and is ultimately determined by the attending physician.

[0031] In the fifth aspect, the present invention provides a method for screening candidate drugs capable of alleviating and / or reducing and / or treating epilepsy, including: Establishing an in vitro neuronal injury cell model, contacting the test substance with the damaged neuronal cells, and measuring whether there is overexpression of lncMCL1 in the neuronal cells after contact. If there is overexpression, the test substance is used as a candidate drug.

[0032] In one embodiment, the disease, disorder or dysfunction associated with loss of neuronal function may be epilepsy or status epilepticus caused by epilepsy.

[0033] In one embodiment, the neuronal injury model may be a hippocampal neuronal injury model. In a specific embodiment, in the hippocampal neuronal injury model, the damaged hippocampal neuronal cells undergo inflammatory necrosis and / or pyroptosis.

[0034] In the present invention, the hippocampal neuronal injury model can be a cell model or an animal model. The cell model can include, but is not limited to, chemical injury models, such as the sodium arsenite injury model; oxidative stress models, such as the hydrogen peroxide model; inflammatory factor stimulation models, such as the lipopolysaccharide and cytokine combined stimulation model; glutamate excitotoxicity models, such as the glutamate and its analogues injury model, and so on. The neuronal cells used in the cell model can be common hippocampal neuronal cell lines, such as the mouse H-22 cell line. The animal model can include, but is not limited to, traumatic brain injury models, such as the fluid percussion injury (FPI) model; ischemia-hypoxia models, such as the bilateral common carotid artery ligation model; chemical injury models, such as the sodium arsenite injury model and the PAPP inhibitor model; inflammatory mediator injury models, such as the LPS-induced model, the experimental autoimmune encephalomyelitis model; gene and protein level regulation models, such as the miRNA regulation model, the protein kinase regulator model, etc.

[0035] In a specific embodiment, the hippocampal neuronal injury model can adopt a glutamate excitotoxicity cell model, that is, hippocampal neuronal cells are treated with glutamate and its analogues (such as kainic acid, NMDA) to induce excitotoxic injury of hippocampal neurons. This model is commonly used to study the neuronal injury mechanism in neurological diseases such as epilepsy and cerebrovascular diseases.

[0036] Therefore, in one embodiment, the screening method as described above includes the following steps: establishing a glutamate excitotoxicity cell model (i.e., a hippocampal neuronal injury model), in the presence of glutamate or its analogue, contacting the test substance with the damaged hippocampal neuronal cells, and measuring whether there is overexpression of lncMCL1 in the damaged hippocampal neuronal cells after contact. If there is overexpression, the test substance is used as a candidate drug.

[0037] In a specific embodiment, the cells used in establishing the glutamate excitotoxicity cell model are hippocampal neuron cell lines, preferably the mouse H-22 cell line. Methods for measuring lncMCL1 overexpression are well known in the art, such as quantitative PCR (qRT-PCR), gene chips, Western blotting, immunohistochemistry techniques, cell function experiments, etc.

[0038] Through in vitro and in vivo experiments, the present invention demonstrates that lncMCL1 can serve as a key regulator of pyroptosis or inflammatory necrosis of epileptic hippocampal neurons, can effectively regulate the pyroptosis or inflammatory necrosis of neuronal cells, and can be used to alleviate, reduce or treat related diseases involving these two mechanisms (such as epilepsy or status epilepticus caused by epilepsy). BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It shows that lncMCL1 is downregulated in neurons of the temporal lobe tissue of patients with temporal lobe epilepsy; among them, A is the heat map analysis result of differential lncRNA and mRNA expression between the temporal cortex (TLE) of patients with temporal lobe epilepsy and the temporal cortex (Control) of patients with traumatic brain injury; B is the functional annotation-based mRNA data enrichment analysis comparing control and TLE cortex; C is the Venn diagram of conserved lncRNAs in humans, mice and rats confirmed by homology analysis; D is the RT-qPCR analysis of lncMCL1 expression in the ipsilateral hippocampus of KA-induced epileptic mice; E is the RT-qPCR analysis of lncMCL1 expression in the hippocampus of PTZ-induced epileptic mice; F is the RT-qPCR analysis of lncMCL1 expression in the ipsilateral hippocampus of KA-induced epileptic rats; G is the violin plot of lncMCL1 expression in the temporal cortex of TLE patients and the temporal cortex of patients with traumatic brain injury; H is the correlation analysis of lncMCL1 expression in the hippocampus with TLE and epilepsy history; I is the RT-qPCR detection of lncMCL1 expression in cytoplasmic and nuclear fractions (n = 3); J is the RNAFISH detection and immunofluorescence staining to examine the localization of lncMCL1 in cortical samples of TLE patients; K is the RNAFISH detection and immunofluorescence staining of NeuN and GFAP to examine the cellular localization of lncMCL1 in the hippocampus of KA-induced epileptic mice; L is the RNAFISH detection to examine the localization and expression of lncMCL1 in the CA1, CA3 and DG regions of the ipsilateral and contralateral hippocampi of KA-induced epileptic mice. The left figure is the panoramic view of the hippocampus, and the right figure is the local view of the CA1, CA3 and DG regions.

[0040] Figure 2Activation of pyroptosis of NLRP3 inflammasome in TLE neurons; among them, A is the mRNA data enrichment analysis based on functional annotation, comparing ov-NC and ov-lncMCL1 HT-22 cells; B is the gene set enrichment analysis showing that high lncMCL1 expression is negatively correlated with the inflammatory response in neurons; C is the immunoblot detection of NLRP3 inflammasome signaling (NLRP3, c-Caspase1, IL1β, and IL18) in the hippocampus of TLE patients and controls; D is the quantification of the corresponding results in C; E is the immunoblot detection of NLRP3 inflammasome signals (NLRP3, c-Caspase1, IL1β, and IL18) in the temporal cortex of TLE patients and controls; F is the quantification of the corresponding results in E; G is the immunofluorescence staining of NLRP3 and NeuN in the hippocampus and temporal cortex of controls and TLE patients; H is the immunofluorescence staining of c-Capase1 and NeuN in the hippocampus and temporal cortex of controls and TLE patients; I is the immunofluorescence staining of NLRP3 and NeuN in the hippocampus of controls and KA-induced epileptic mice (TLE mice).

[0041] Figure 3 Overexpression of lncMCL1 in vitro inhibits glutamate-induced pyroptosis of hippocampal neurons; A is the RT-qPCR analysis of the expression of lncMCL1 in HT-22 cells treated with different concentrations of glutamate (mean ± SEM, n = 3, one-way ANOVA); B is the IF staining of NLRP3 and c-Caspase1 in control and Glu-treated HT-22, Scarlbar is 50 μm; C is the immunoblot detection of NLRP3 inflammasome signaling (NLRP3, c-Caspase1, IL1β, and IL18) in control and Glu-treated HT-22; D is the quantification of the corresponding results in C, (mean ± SEM, n = 4 - 5, student t-test); E is the immunoblot detection of the pyroptosis marker in control and Glu-treated HT-22 transfected with ov-NC or ov-lncMCL1; F is the quantification of the corresponding results in E, (mean ± SEM, n = 2 - 3, two-way ANOVA); G is the ELISA detection of the concentration of IL18 / IL1β in the culture medium of control and Glu-treated HT-22 transfected with ov-NC or ov-lncMCL1, (mean ± SEM, n = 3, two-way ANOVA); H is the IF staining of NLRP3 in control and Glu-treated HT-22 transfected with ov-NC or ov-lncMCL1; I is the IF staining of c-Caspase1 in control and Glu-treated HT-22 transfected with ov-NC or ov-lncMCL1.

[0042] Figure 4Knockdown of lncMCL1 in vitro promotes glutamate-induced pyroptosis in HT-22 cells; among them, A is RT-qPCR analysis of lncMCL1 expression to evaluate the siRNA interference efficiency, (mean ± SEM, n = 3, one-way ANOVA); B is immunoblotting to detect pyroptosis markers in HT-22 transfected with NC or siRNA with or without Glu treatment; C is the quantification of the corresponding results in B, (mean ± SEM, n = 3, two-way ANOVA); D is ELISA to detect the concentration of IL18 / IL1β in the culture medium of HT-22 transfected with NC or siRNA with or without Glu treatment; E is IF staining of NLRP3 in HT-22 transfected with NC or siRNA with or without Glu treatment; F is IF staining of c-Caspase1 in control and Glu-treated HT-22 (transfected with NC or siRNA).

[0043] Figure 5 Knockout of lncMCL1 by CRISPR / Cas9 promotes glutamate-induced pyroptosis in HT-22 cells; among them, A is a schematic diagram of knocking down lncMCL1 using the CRISPR / Cas9 system; B is the sequencing result confirming the insertion of lncMCL1 by CRISPR / Cas9 in HT-22 cells; C is immunoblotting to detect pyroptosis markers in Cas9-NC and Cas9-lncMCL1 HT-22 with or without Glu; D is the quantification of the corresponding results in D, (mean ± SEM, n = 3, two-way ANOVA).

[0044] Figure 6 In vivo verification that lncMCL1 inhibits pyroptosis of epileptic hippocampal neurons; among them, A is the timeline of the experimental design; B is the number of SRSs of epileptic mice with or without overexpression of lncMCL1 from day 28 to day 35 after SE by video monitoring; C is the violin plot of the total 7-day epileptic seizures induced by KA in the control group and the lncMCL1 overexpression group; D is the EEG time-frequency diagram showing the brain wave changes of PTZ-induced mice in the control group and the lncMCL1 overexpression group; E is the violin plot of the total 7-day epileptic seizures induced by PTZ in the control group and the lncMCL1 overexpression group; F is Nissl staining to examine the number of neurons in the ipsilateral and contralateral hippocampal CA1 and CA3 regions of TLE mice. The left figure is the panoramic view of the hippocampus, and the right figure is the CA1 and CA3 regions; G is the quantification of the number of neurons. Three positions were selected for analysis from each region, (mean ± SEM, n = 3, one-way ANOVA); H is the immunofluorescence staining of NLRP3, c-Capase1, NeuN, and GFAP in the hippocampus of control and TLE mice.

[0045] The above data are shown as the mean ± SEM of independent experiments. *** indicates P < 0.001, ** indicates P < 0.01, * indicates P < 0.05, and ns indicates no significant difference. Detailed implementation mode

[0046] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention, and they should not be construed as limiting the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for the purpose of description and cannot be construed as indicating or implying relative importance.

[0047] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0048] The male C57 BL / 6 mice (weighing 18 g - 20 g) and Sprague-Dawley rats (weighing 200 g - 220 g) used in the following embodiments were from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in a constant temperature room, raised according to a standard 12-hour light / dark cycle, and had free access to standard rodent feed and water. All animal experiment procedures were approved by the Ethics Committee of Beijing Tiantan Hospital, Capital Medical University (Ethics Nos. AEEI-2018-200 and AEEI-2019-097).

[0049] The mouse neuronal cell line HT-22 and HEK-293T cells (derived from ATCC (American Type Culture Collection)) were cultured in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (both from Gibco, Grand Island, NY, USA) at 37°C and 5% CO2. For glutamate treatment, the cells were incubated in DMEM medium containing 5 mM glutamate for 24 hours. For IL1β treatment, the cells were incubated in DMEM medium containing 75 and 100 ng / ml IL1β for 6 hours and 24 hours respectively. For IL18 treatment, the cells were incubated in DMEM medium containing 75 and 100 ng / ml IL18 for 6 hours and 24 hours respectively.

[0050] The nucleotide sequences involved in the following examples are shown in Table 1:

[0051] Note: In Table 1, the bold and underlined part in the sequence corresponding to SEQ ID NO:1 is the target sequence of sgRNA.

[0052] CRISPR-mediated knockout of the lncMCL1 gene in the mouse HT-22 cell line The target sequence of lncMCL1 sgRNA was designed by Hanheng Biotechnology (Shanghai) Co., Ltd. To prepare CRISPR lentivirus, HEK293T cells were seeded in a 100 mm culture dish and co-transfected with 10 μg of lentiCRISPRv2-gRNA or lentiCRISPRv2 control plasmid (Addgene, MA, USA, plasmid #52961), 10 μg of psPAX2 plasmid and 5 μg of PMD2G plasmid using the Lipofiter TM transfection system. The culture supernatants containing lentivirus were collected at 48 hours and 72 hours after transfection and used to infect HT-22 cells for 16 - 24 hours. After one week, a polyclonal KO cell line was obtained, and then screened through a 96-well plate for 1 - 2 months. Finally, a monoclonal cell line with stable target gene knockout was obtained.

[0053] RNA sequencing and data analysis HT-22 cells transfected with 80MOI LV-MCL1 virus were collected for RNA sequencing. RNA-seq was used to detect the mRNA expression profile of HT-22 cells with lncMCL1 overexpression using HiSeq3000 (Illumina) at a Shanghai biotechnology company. HISAT was used for transcriptional level expression analysis of the RNA-seq experiment and reference annotation was performed to generate FPKM values of known gene models. edgeR was used to identify differentially expressed genes. The fold change was also estimated based on the FPKM in each sample. Differentially expressed genes were selected using the following filtering criteria: FDR ≤ 0.05 and fold change ≥ 2.

[0054] RNA extraction and RT-qPCR Total RNA was extracted using TRIzol reagent (Invitrogen), and cDNA was obtained using MonScript™ RT II All-in-One Mix (Monad) according to the manufacturer's instructions, unless otherwise specified. RT-qPCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme), and the products were quantified using a CFX Connect real-time PCR detection system (Bio-Rad). The relative gene expression levels were normalized to the level of GAPDH and calculated using the 2 -ΔΔCt method.

[0055] Immunofluorescence (IF) staining Cells seeded on coverslips or cryosectioned mouse brains were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 5 minutes, and blocked with QuickBlock™ Immunostaining Blocking Buffer (Beyotime) for 1 hour at room temperature. Then, the cells or brain tissues were incubated overnight at 4°C with the following primary antibodies: anti-NLRP3 (Abcam, ab214185, 1:100), anti-Cleaved-Caspase-1 (Cell Signaling Technology, 89332, 1:100), anti-NeuN (Abcam, ab1024224, 1:300), anti-GFAP (Abcam, ab53554, 1:500). The next day, the cells or brain tissues were incubated with the corresponding fluorescent secondary antibodies (Proteintech, SA00013-2, SA00013-3, and SA00013-4).

[0056] Western blot analysis Each group of brain tissues or cells was homogenized on ice with RIPA buffer (Beyotime Biotechnology, China) supplemented with 1:1000 protease inhibitor mixture and 1:1000 phosphatase inhibitor mixture (MedChemExpress). After quantification using a BCA kit (ThermoFisher Scientific), equal amounts of cell lysate or brain tissue lysate proteins were added to SDS-PAGE sample loading buffer (Beyotime), boiled, denatured, separated by SDS-PAGE, and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the membrane was blocked with 5% non-fat milk dissolved in TBST and incubated overnight with the primary antibody, followed by incubation with the secondary antibody and exposure to Immobilon Crescendo Western HRP substrate (Millipore). The primary antibodies used were as follows: anti-NLRP3 (Abcam, ab210491, 1:800), anti-pro-Caspase1 (Abcam, 179515 and ab1872, 1:500), anti-Cleaved Caspase-1 (Cell Signaling Technology, 89332, 1:1000), anti-IL1β (Abcam, ab9722, 1:500), anti-IL18 (Proteintech, 10633-1-AP, 1:500), anti-GSDMD (Abcam, ab209845, 1:500), anti-GSDMD-N (CST, 50928, 1:1000), anti-ACS (Cell Signaling Technology, 67873, 1:500), anti-TNFα (Abcam, ab6671, 1:500), anti-GAPDH (Abcam, ab181602, 1:3000). The secondary antibodies were: anti-rabbit IgG (Cell Signaling Technology, 7074, 1:2000) and anti-mouse IgG (Cell Signaling Technology, 7076, 1:2000).

[0057] Fluorescence in situ hybridization Deparaffinize, dehydrate the paraffin-embedded sections (4-μm thick) and treat them with 1 M sodium thiocyanate. Then digest the sections in pepsin solution, fix them in 4% formaldehyde, dehydrate them by sequentially immersing them in 70%, 85% and 100% ethanol, and then air-dry. Incubate the sections with digoxigenin (DIG)-labeled probes (Probes 1-3 shown in Table 1), and then incubate them with the DyLight 594-conjugated IgG fraction (Abcam, ab96873) conjugated with monoclonal mouse anti-DIG antibody (Abcam, ab116590). Counterstain the cell nuclei with DAPI. Scan and analyze the sections as described above.

[0058] Immunoprecipitation (IP) The IP assay was performed using the Pierce™ Classic Magnetic IP / Co-IP Kit (Thermo Fisher Scientific). Briefly, cells were incubated with IP lysis / wash buffer on ice for 5 minutes with regular mixing. The cell lysate was bound to the IP antibody to prepare a 500 μL antibody / lysate solution, and the solution was incubated overnight at 4 °C. Then, the solution was incubated with 25 μL Pierce Protein A / G magnetic beads at room temperature for 1 hour. The collected bead-protein complexes were heated to 96-100 °C in Lane Marker sample buffer (Thermo Fisher Scientific) for 10 minutes and then subjected to SDS-PAGE.

[0059] RNA pull-down assay and mass spectrometry The RNA pull-down assay was performed using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the manufacturer's protocol. Mouse lncMCL1 sequences were transcribed in vitro using a biotin RNA labeling mix and T7 RNA polymerase (Invitrogen). Then the biotinylated lncMCL1 RNA was incubated with streptavidin-conjugated magnetic beads and the total lysate of HT-22 cells at room temperature for 2 hours. After incubation, the RNA-protein complexes bound to the beads were washed four times with wash buffer. Then the proteins were eluted and resuspended in protein lysis buffer. Finally, the eluted proteins were analyzed by SDS-PAGE and subjected to mass spectrometry once.

[0060] RNA immunoprecipitation assay The RNA immunoprecipitation (RIP) assay was performed using an RNA immunoprecipitation (RIP) kit (BersinBio) according to the manufacturer's instructions. Briefly, 2×107 HT-22 cells were harvested and lysed with polysome lysis buffer. The cell extracts were co-immunoprecipitated with anti-DDX3X antibody (Santacruz, sc-365768) or anti-NEDD4 (Proteintech, 83112-1-RR), and the recovered RNA was analyzed by qRT-PCR using lncMCL1 primer pairs. Total RNA (input control) and normal mouse IgG control were measured simultaneously to confirm that the detected signal was from RNA specifically bound to DDX3X or NEDD4.

[0061] Nissl staining Mice were perfused transcardially with 0.01 M phosphate buffer (PBS; pH 7.4), and then with 4% paraformaldehyde in 0.01 M PBS. The brains were fixed overnight at 4°C with 4% paraformaldehyde in 0.01 M PBS, and then placed in 30% sucrose solution until the brains sank to the bottom of the chamber. The optimal cutting temperature (OCT)-embedded tissues were cut into 25 μm-thick sections and mounted on pre-coated glass slides (GoldenBridge, Shanghai, China). The slides were stained with cresyl violet (Beyotime Institute of Biotechnology, Shanghai, China) for 10 minutes. Then the slides were rinsed with distilled water, dehydrated with ethanol at different concentrations (70%, 80%, 90%, 100%), cleared with xylene, and coverslipped with neutral balsam. Neurons in the hippocampal CA3 and CA1 regions were analyzed. The average number of neurons in three 40× light fields at the same section position of each mouse was calculated.

[0062] Statistical analysis All statistical analyses were performed using GraphPad Prism software (version 7.0; GraphPad Software, USA). Student's t-test or Wilcoxon Mann-Whitney test was used to compare data between two different groups. One-way or two-way analysis of variance (ANOVA) was used to analyze statistical comparisons among multiple groups, followed by appropriate post hoc tests. Data were expressed as mean ± standard error of the mean (SEM). P < 0.05 was considered significant.

[0063] Example 1. Screening of lncRNA expression profiles in the temporal cortex of TLE patients Temporal lobe tissues of patients with temporal lobe epilepsy (TLE) were collected for microarray analysis, and the temporal lobe cortex of patients who underwent surgical resection after traumatic brain injury was used as the control group (n = 4). First, RNA samples were reverse transcribed into cDNA, and then these cDNA samples were labeled and subjected to array hybridization according to the Agilent one-color microarray gene expression analysis protocol (Agilent Technologies, Santa Clara, CA, USA). When the fold change (FC) ≥ 2 and P < 0.05, differential expression of lncRNA and mRNA was considered to exist between the two groups of samples. The analysis results are as Figure 1 shown in A. Among them, 490 differentially expressed mRNAs were found between the TLE patient group and the control group, of which 287 were up-regulated and 203 were down-regulated; 736 differentially expressed lncRNAs were found, of which 457 were up-regulated and 279 were down-regulated.

[0064] The above differentially expressed genes were put into the Database for Annotation, Visualization and Integrated Discovery (DAVID; http: / / david.abcc.ncifcrf.gov / ), and GO was used to identify the molecular functions represented in the gene profile. As Figure 1 shown in B, functional annotation and pathway analysis showed that the differentially expressed genes were involved in a variety of functions related to inflammatory responses, such as positive regulation of chemokine production, positive regulation of interleukin 8 production, positive regulation of acute inflammatory responses, etc., suggesting that neuroinflammation is one of the important pathological processes in the occurrence of epilepsy.

[0065] Since lncRNAs are different from coding genes, miRNAs and circRNAs and lack evolutionary conservation, while highly conserved genes often play important roles in maintaining the normal biological functions of organisms. Therefore, Blastn homology analysis was used to perform homology analysis on 736 differentially expressed lncRNAs (e value less than 1e-5) to identify lncRNAs conserved among species. The results are as Figure 1 shown in C. The results showed that 140 lncRNAs were conserved between humans and mice (Mus musculus), 63 lncRNAs were conserved between humans and rats (Rat), and 5 lncRNAs were conserved among humans, rats and mice, which were lnc-MCL1-2:1 (NONHSAT006310.2, abbreviated as lncMCL1), NR_002139, ENST00000501122, ENST00000562284 and ENST00000605417 respectively.

[0066] 2. lncMCL1 is down-regulated during the occurrence of epilepsy 2.1. Construction of a seizure model induced by intra-amygdala injection of kainic acid (KA): Inject 0.3 μL of KA (1 μg / μL, Sigma-Aldrich, St. Louis, Missouri, USA) into the right amygdala of mice (0.94 mm posterior to bregma, 2.75 mm lateral, 4.75 mm depth), and inject 0.7 μL into the right amygdala of rats (2.76 mm posterior to bregma, 4.5 mm lateral, 8.6 mm depth). The sham-operated control group received an intra-amygdala injection of the same volume of normal saline. Cortical electroencephalogram recording electrodes were implanted in the mice, and continuous video electroencephalogram monitoring (24 hours / day) was performed on the spontaneous recurrent seizures (SRS) of the epileptic mice. The SRS was scored according to the Racine scale. Racine scale: grade 0, no seizure; grade I, facial myoclonus, piloerection, and scratching and climbing; grade II, grade I behavior accompanied by head nodding; grade III, grade II behavior accompanied by forelimb clonus; grade IV, grade III behavior accompanied by hindlimb clonus or tetanus; grade V, grade IV behavior accompanied by continuous restlessness and rolling. Only the SRS frequencies of animals in stage IV and V were recorded and analyzed. Any level of seizure detected by video electroencephalogram monitoring indicated successful model establishment. Subsequently, on days 1, 10, and 30 after KA injection, the expression patterns of lncMCL1 in the hippocampal tissues of mice and rats were analyzed. The analysis results are shown in Figure 1 D and F as follows. It can be seen that in the mouse (Mouse) and rat (Rat) seizure models, the expression of lncMCL1 was downregulated during epileptogenesis.

[0067] 2.2. Construction of the pentylenetetrazol (PTZ) model: Cortical electroencephalogram recording electrodes were implanted in the mice, and 35 mg / kg of PTZ was injected intraperitoneally every other day. The epileptic behavior was monitored by video electroencephalogram for 30 minutes after each injection of PTZ. The seizure stage was scored as follows: 0: normal behavior, no abnormality. 1: immobile, prone. 2: head nodding, facial, forelimb, or hindlimb myoclonus. 3: persistent generalized myoclonus, myoclonic jerks, tail rigidly raised. 4: upright, tonic seizure, lateral fall. 5: tonic-clonic seizure, fall on the back, running wild, and jumping. 6: death. To produce fully kindled animals, once the animal experienced a seizure scored as 5 (tonic-clonic seizure), the injection was completed within the next three administrations. To evaluate the vulnerability of mice injected with AAV to PTZ, 35 mg / kg was injected into each mouse every other day for a total of 6 times. Any level of seizure detected by video electroencephalogram monitoring indicated successful model establishment. After injection of PTZ, the expression patterns of lncMCL1 in the hippocampal tissues of the mice were analyzed. The analysis results are shown in Figure 1As shown in E, in a pentylenetetrazol (PTZ)-induced mouse epilepsy model, the expression of LncMCL1 in the hippocampus was also significantly decreased.

[0068] 2.3. The analysis of lncMCL1 expression was extended to 20 TLE patient samples, and the analysis results are as Figure 1 shown in G. It can be seen that the expression of lncMCL1 was significantly decreased in TLE patients.

[0069] 2.4. A correlation analysis was performed on the expression of lncMCL1 in the hippocampal tissues of TLE patients and the epilepsy history of the patients. The results are as Figure 1 shown in H. It can be seen that the expression of lncMCL1 in the hippocampal samples of TLE patients was negatively correlated with the epilepsy history.

[0070] In summary, these results indicate that the conserved lncMCL1 is decreased in epilepsy and may play an important role in the occurrence of epilepsy.

[0071] 2.5. To further determine the subcellular localization of lncMCL1, according to the manufacturer's instructions, NE-PER nuclear and cytoplasmic extraction reagents (#78833, Pierce, Rockford, IL) were used to isolate and purify the cytoplasmic and nuclear RNA of HT-22 cells, and then qRT-PCR was performed. The results are as Figure 1 shown in I, indicating that lncMCL1 is mainly located in the cytoplasm.

[0072] 2.6. Paraffin-embedded sections of the cortical samples of TLE patients and the hippocampal tissues of KA-induced mouse and rat epilepsy models were prepared and subjected to fluorescence in situ hybridization (FISH) analysis; specifically, the paraffin-embedded sections (4 μm thick) were dewaxed, dehydrated and treated with 1 M sodium thiocyanate. Then the sections were digested in pepsin solution, fixed in 4% formaldehyde, dehydrated by soaking in 70%, 85% and 100% ethanol in sequence, and then air-dried. The sections were incubated with a digoxigenin (DIG)-labeled probe 1-3, and then incubated with a DyLight 594-conjugated IgG fraction (Abcam, ab96873) that was conjugated with a monoclonal mouse anti-DIG antibody (Abcam, ab116590). The cell nuclei were counterstained with DAPI. The results are as Figure 1 shown in J-K, which indeed revealed the distribution of lncMCL1 in the cytoplasm and a decrease in the expression of lncMCL1 in the ipsilateral and contralateral hippocampi of epileptic mice injected with KA. Double staining of lncMCL1 and the astrocyte protein marker GFAP in the brain samples of epileptic mouse and rat models both indicated that lncMCL1 was mainly located in the cytoplasm of neurons rather than astrocytes.

[0073] 2.7. RNA FISH detection of lncMCL1 in the ipsilateral and contralateral hippocampal CA1, CA3, and DG regions of KA-induced epileptic mice. The results are shown in Figure 1 Figure L. The expression of lncMCL1 was decreased in the ipsilateral and contralateral hippocampi of epileptic mice injected with KA ( Figure 1 K, L). Double staining of lncMCL1 and the astrocyte protein marker GFAP in brain samples of epileptic mouse and rat models showed that lncMCL1 was mainly located in the cytoplasm of neurons rather than astrocytes ( Figure 1 K).

[0074] 3. Activation of NLRP3 inflammasome-mediated pyroptosis in neurons of epileptic tissues.

[0075] To explore the biological function of lncMCL1, lncMCL1 was overexpressed in HT-22 cells, and then RNA-Seq was used to screen transcriptome RNA. After overexpression of lncMCL1 in HT-22 cells, 103 RNAs were upregulated and 246 RNAs were downregulated (ov-NC vs ov-lncMCL1, q-value ≤ 0.05, |Fold-change| ≥ 2). Functional annotation, pathway analysis, and GSEA analysis showed that the differentially expressed genes were involved in many functions and pathways related to inflammatory responses (GO terms: cellular response to interleukin-1, chemokine activity, chemokine receptor binding), indicating that lncMCL1 is related to neuronal inflammation ( Figure 2 A - B).

[0076] Pyroptosis is an inflammatory programmed cell death triggered by various pathological stimuli. This process activates inflammasomes, leading to the maturation and release of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-18 (IL-18). Given that pyroptosis occurs in neurons in epilepsy and other neurological diseases, and we found that the biological function of lncMCL1 in neurons is related to the above interleukin-1, it is speculated that lncMCL may be related to pyroptosis.

[0077] Then, NLRP3 inflammasome-mediated neuronal pyroptosis proteins, including NLRP3, Caspase1, IL1b, and IL18, were detected in temporal lobe epilepsy. Western blot analysis showed that compared with the control group, the hippocampus of TLE ( Figure 2The protein levels of NLRP3, cleaved-Caspase1 (c-Caspase1), IL1β, and IL18 were significantly increased in the hippocampus (C, D) and temporal cortex Figure 2 (E, F). Double immunofluorescence staining was used to co-localize NLRP3 and c-Caspase1 with the neuronal marker NeuN and the astrocyte marker GFAP. The results further showed that NLRP3 and c-Caspase1 were mainly localized in neurons and astrocytes in the hippocampus and temporal cortex of TLE patients Figure 2 (G, H), as well as in the hippocampus at different stages of epileptogenesis in the KA-induced TLE mouse model Figure 2 (I), indicating that the NLRP3 inflammasome is activated in TLE.

[0078] Example 2: In vitro experiments confirmed that lncMCL1 inhibits pyroptosis of epileptic hippocampal neurons This example explored the role of lncMCL1 in glutamate (Glu)-induced hippocampal neuronal injury and its relationship with the inflammatory necrosis (pyroptosis) caused by status epilepticus (SE).

[0079] 1. An in vitro damaged neuron model was established by treating HT22 (mouse hippocampal neuronal cell line) with glutamate. The specific method was as follows: The cells were incubated in DMEM medium containing 5 mM glutamate for 24 hours. The expression level of LncMCL1 in the HT22 cell line was detected. The results were as Figure 3 shown in A. Consistent with the in vivo experimental results, excessive glutamate significantly downregulated the expression level of lncMCL1 in the cell line. Through Western blot and immunofluorescence (IF) staining experiments, the protein and mRNA expression levels of NLRP3, GSDMD-FL, GSDMD-N, pro-Caspase1, and c-Caspase1 in the HT22 mouse hippocampal neuronal cell line were detected. The results were as Figure 3 shown in B-D. It can be seen that compared with the control group, the expression levels of NLRP3, GSDMD-FL, GSDMD-N, pro-Caspase1, and c-Caspase1 in the cells were significantly increased after glutamate treatment, indicating that glutamate can induce inflammatory necrosis of neurons.

[0080] 2. Insert the coding sequence of lncMCL1 (SEQ ID NO:1) into the lentiviral vector (pHBLV-CMV-MCS-EF1-Zsgreen1-T2A-puro). The GV358 viral vector carrying the scrambled sequence was used as a negative control (NC).

[0081] Seed HT-22 cells into a 6-well plate at a density of 3×10 5 cells / well. When the cell confluence reaches 30%-50%, transfect at 80 MOI, and add 1 ml of complete medium containing 40 μl of Hitrans GP infection reagent and 20 μl of NC lentiviral vector, 20 μl of LV-MCL1. At 12-16 h after infection, replace the medium containing the lentiviral vector with the medium containing fresh serum. Two days after infection, screen the cells with 3 μg / ml puromycin. After the screening is completed, treat the cells with PBS buffer and glutamate at a concentration of 5 mM for 24 hours. Then, analyze the expression levels of proteins related to the NLRP3 inflammasome signaling pathway, including NLRP3, ASC, c-Caspase1, GSDMD-N, TNFα, IL-1β, and IL-18, by Western blot, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) experiments. The analysis results are shown in Figure 3 E-I. The results showed that overexpression of lncMCL1 significantly inhibited the expression of proteins related to the NLRP3 inflammasome signaling pathway under glutamate treatment conditions, while no significant changes were observed under normal conditions.

[0082] 3. To verify the mechanism of action of lncMCL1, small interfering RNA (siRNA) technology was further used to knockdown lncMCL1. First, synthesize small interfering RNAs (siRNA1-3) against mouse lncMCL1 and scrambled control siRNA. Transfect 20 nmol of lncMCL1 siRNA or scrambled siRNA into HT-22 cells using Lipofectamine RNAiMAX (Invitrogen). After 8 hours of transfection in antibiotic-free medium, update the cells with normal medium. Collect the cells for analysis 48 hours after transfection. The analysis results are shown in Figure 4 A. siRNA1 and siRNA2 (but not siRNA3) were able to significantly reduce the expression level of lncMCL1. Using the same analysis method as the overexpression experiment, analyze the cells with reduced lncMCL1 expression level. The results are shown in Figure 4As shown in B-E, it can be seen that knocking down lncMCL1 using siRNA1 and siRNA2 significantly upregulated the expression of proteins related to the NLRP3 inflammasome signaling pathway and enhanced the levels of molecular markers of inflammatory necrosis.

[0083] 4. As Figure 5 As shown in A-B, a monoclonal cell line with lncMCL1 knockout was constructed using the CRISPR-Cas9 gene editing technology. Meanwhile, the monoclonal cell line with lncMCL1 knockout was treated with a glutamate solution at a concentration of 5 mM. At the same time, using PBS buffer as a control, after 24 hours of treatment, Western blot analysis was performed on the proteins related to pyroptosis in the monoclonal cell line with lncMCL1 knockout. The analysis results are as Figure 5 As shown in D-E, it can be seen that Cas9-mediated lncMCL1 knockout and siRNA-mediated lncMCL1 knockdown showed consistency at the molecular level.

[0084] Example 3. In vivo experiments confirmed that lncMCL1 inhibits pyroptosis of epileptic hippocampal neurons 1. The in vivo biological function of lncMCL1 was studied by overexpression using an adeno-associated virus (AAV) delivery system. As Figure 6 As shown in A, the cDNA sequence of lncMCL1 (ENSMUST00000175032, SEQ ID NO: 1) was cloned into the pHBAAV-CMV-MCS-EF1-mcherry AAV expression vector of Shanghai Hanheng Biotechnology Co., Ltd. to construct a recombinant AAV carrying lncMCL1 driven by the CMV promoter (AAV-MCL1). The cDNA sequence of DDX3X (NM_010028.3) was cloned into the CMV enhancer-MCS-3Flag-FT2A-GFP-WPRE-BGH polyA AAV expression vector of Shanghai GeneChem Co., Ltd. to construct a recombinant AAV carrying DDX3X driven by the CMV promoter (AAV-DDX3X). The titers of these AAV vectors were diluted to 5×10 12 vg / ml. A total of 1 μL of AAV-MCL1 vector or AAV-DDX3X was injected into the right dorsal hippocampus (2.5 mm posterior to bregma, 2.1 mm lateral, 1.9 mm deep). Two weeks after AAV injection, KA was given to induce epilepsy, and the frequency of chronic seizures in mice with or without lncMCL1 overexpression was monitored using video surveillance from the 5th to the 6th week after KA-induced epilepsy. The results are as Figure 6 As shown in B-E, compared with sham-operated mice, the frequency of recurrent seizures in lncMCL1 overexpressing mice was significantly reduced.

[0085] 2. Nissl staining was performed on the hippocampal tissues of mice overexpressing lncMCL1 (ov-lncMCL1) and their control mice (ov-NC). The results are shown in F-G of Figure 6 Figure, indicating that pretreatment with ov-lncMCL1 can protect hippocampal neurons from SE-induced damage in the ipsilateral CA3 and CA1 regions and the contralateral CA1 and CA3 regions.

[0086] IF analysis showed that, as shown in Figure 6 Figure H, pretreatment with ov-lncMCL1 reduced the protein levels of NLRP3, c-Caspase1, and GSDMD-N.

[0087] Based on the above in vivo and in vitro experimental results, during epileptogenesis, the decrease in lncMCL1 loses its inhibitory effect on the NLRP3 inflammasome, leading to pyroptosis of nerve cells and epileptic seizures.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features. However, such modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a substance that enhances the expression of lncMCL1 in neuronal cells in the preparation of a drug for relieving and / or treating diseases, disorders or dysfunctions associated with neuronal function deficiency.

2. The application according to claim 1, wherein The substance that enhances the expression of lncMCL1 in neuronal cells is a biomaterial related to the lncMCL1, and the biomaterial is selected from at least one of A1)-A4): A1) A nucleic acid molecule encoding the lncMCL1; A2) An expression cassette containing the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2); A4) A recombinant microorganism containing the nucleic acid molecule described in A1), or a recombinant microorganism containing the expression cassette described in A2), or a recombinant microorganism containing the recombinant vector described in A3).

3. The application according to any one of claims 1-2, characterized in that, The diseases, disorders or dysfunctions associated with neuronal function deficiency are epilepsy or status epilepticus caused by epilepsy.

4. The application according to claim 1, wherein The neuronal cells are hippocampal neuronal cells.

5. The application according to claim 1, wherein The neuronal cells are derived from the hippocampal tissue or the cerebral cortex.

6. A method for in vitro regulating the degree of neuronal pyroptosis or inflammatory necrosis, characterized in that, Including regulating the expression of lncMCL1 in neuronal cells by the substance that enhances the expression of lncMCL1 in neuronal cells according to any one of claims 1-5 in vitro to regulate the degree of pyroptosis or inflammatory necrosis of the neuronal cells.

7. The method according to claim 6, wherein The regulation results in a decrease in the expression of proteins related to the NLRP3 inflammasome signaling pathway and / or a decrease in the content of molecular markers of inflammatory necrosis in the regulated neuronal cells compared with the unregulated neuronal cells.

8. A pharmaceutical composition, characterized in that, Including a substance that enhances the expression of lncMCL1 in neuronal cells and a pharmaceutically acceptable carrier.

9. A method for screening a candidate drug capable of alleviating and / or reducing and / or treating a disease, disorder or dysfunction associated with neuronal loss of function, characterized in that, Including: Establishing an in vitro neuronal injury cell model, contacting a test substance with the damaged neuronal cells, and measuring whether there is overexpression of lncMCL1 in the damaged neuronal cells after the contact. If there is overexpression, the test substance is used as a candidate drug.

10. The method according to claim 9, characterized in that The damaged neuronal cells undergo inflammatory necrosis and / or cell pyroptosis.

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