Use of lncmcl1 in regulating the degree of neuron cell pyroptosis and alleviating and treating related diseases

Abstract

The application provides application of lncMCL1 in regulation of pyroptosis degree of neuron cells and alleviation and treatment of related diseases, and relates to the technical field of biological medicine.The application specifically discloses regulation of pyroptosis of neuron cells by enhancing or reducing expression of lncMCL1, and further discloses application of a substance for regulating lncMCL1 expression enhancement in preparation of related diseases of neuron function loss, especially epilepsy.The application is verified by in-vivo experiments and in-vitro experiments, and lncMCL1 can be used as a key regulation factor of pyroptosis of neuron cells by regulating inflammatory response of neuron cells, and can effectively regulate pyroptosis of neuron cells and alleviation and treatment of related diseases of neuron function loss.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine technology, and in particular to the application of lncMCL1 in regulating the degree of neuron cell pyroptosis and relieving and treating related diseases. BACKGROUND

[0002] Epilepsy is a common neurological disorder affecting 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 fully control seizures and eventually develop drug-resistant epilepsy. Temporal lobe epilepsy (TLE) is one of the most common intractable 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 cell self-destruction, which can be triggered by cell stress and inflammatory response. PCD includes various forms of cell death, such as apoptosis, autophagy and programmed necrosis, which is further divided into necroptosis, ferroptosis and pyroptosis. Pyroptosis is a well-known pro-inflammatory form of programmed cell death, which is associated with epilepsy in both animal models and patients with epilepsy. Pyroptosis is a PCD triggered by immune cytokine activation, 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 (e.g. IL-1β, IL-18) and cell membrane rupture; and the non-classical (caspase-1 independent) pathway, involving caspases such as Caspase-11, Caspase-4 or Caspase-5. Clinical and animal studies have shown that the expression of NLRP3, caspase-1 and IL-1β is increased in the hippocampal tissue of TLE patients. Notably, pyroptosis mainly occurs in hippocampal neurons, usually triggered by abnormal activation of NLRP3 inflammasome. Recent studies have shown that knocking out NLRP3 can inhibit the loss of CA3 neurons and the expression of IL-1β caused by epilepsy, suggesting that the classical pyroptosis pathway dependent on NLRP3 inflammasome plays an important role in the loss of epileptic neurons. Studies have shown that factors such as oxidative stress, transient receptor potential channels regulate the activation of NLRP3 and pyroptosis in epilepsy. In addition, the ubiquitin-proteasome pathway and inflammation-related pathway initiators such as gut-enriched Kruppel-like factor, signal transducer and activator of transcription 3 (STAT3) may also affect the function of NLRP3.

[0004] A large amount of evidence shows that lncRNA is a key regulator of NLRP3 inflammasome and pyroptosis. The regulation mediated by lncRNA is related to its subcellular localization, and cytoplasmic lncRNA plays a role in neuronal loss and inflammatory response in epilepsy; the regulation process includes activation of NLRP3 inflammasome, neuronal apoptosis, and overactivation of astrocytes and microglia. Therefore, seeking effective lncRNA targets that can regulate neuronal pyroptosis and treat epilepsy is one of the technical problems to be solved by those skilled in the art. SUMMARY

[0005] The application provides application of lncMCL1 in regulating degree of neuron cell pyroptosis and treatment of epilepsy, and lncMCL1 can be used as a new target to regulate degree of neuron cell pyroptosis and treatment of epilepsy.

[0006] In a first aspect, the application provides use of a substance for enhancing expression of lncMCL1 in a neuron cell in preparation of a medicament for alleviating and / or treating a disease, disorder or dysfunction related to loss of neuron function.

[0007] In the use as described above, the substance for enhancing expression of lncMCL1 in a neuron cell is a biological material related to the lncMCL1, and the biological material is selected from at least one of A1) to A4):

[0008] A1) a nucleic acid molecule encoding the lncMCL1;

[0009] A2) an expression cassette containing the nucleic acid molecule of A1);

[0010] A3) a recombinant vector containing the nucleic acid molecule of A1), or a recombinant vector containing the expression cassette of A2);

[0011] A4) a recombinant microorganism containing the nucleic acid molecule of A1), or a recombinant microorganism containing the expression cassette of A2), or a recombinant microorganism containing the recombinant vector of A3).

[0012] In the use as described above, the neuron cell is derived from at least one of hippocampal tissue and cerebral cortex.

[0013] In the use as described above, the neuron cell is derived from a human or a non-human primate. Further, the non-human primate can be at least one of a rat and a mouse.

[0014] In a specific embodiment, the neuron cell is a hippocampal neuron cell.

[0015] In a specific embodiment, when the neuron cell is derived from a mouse hippocampal tissue, the nucleic acid molecule of A1) is a cDNA molecule or a DNA molecule having a coding sequence of a coding strand with a sequence as shown in SEQ ID NO: 1.

[0016] In the use as described above, the disease, disorder or dysfunction related to loss of neuron function is epilepsy. Further, the epilepsy is temporal lobe epilepsy. Still further, the disease, disorder or dysfunction related to loss of neuron function can be status epilepticus caused by epilepsy.

[0017] The application as described above enhances the expression of lncMCL1 in the neuronal cells by overexpression. In one embodiment, the coding sequence of lncMCL1 can be introduced into an overexpression vector to obtain a recombinant vector containing the coding sequence of lncMCL1, and then the recombinant vector containing the coding sequence of lncMCL1 is injected into the hippocampal tissue to enhance the expression of lncMCL1 in the neuronal cells of the hippocampal tissue.

[0018] In one embodiment, the overexpression vector can be an adeno-associated virus (AAV) vector or a lentivirus vector that can infect neuronal cells or animal models.

[0019] The application as described above alleviates and / or treats the disease, disorder or dysfunction related to the loss of neuronal function, which is manifested as at least one of the following: a decrease in the frequency of seizures, protection of neuronal cells in the hippocampal tissue from damage, and a decrease in the expression of NLRP3 inflammasome signaling pathway-related proteins and / or the content of molecular markers of inflammatory necrosis in the neuronal cells.

[0020] In a second aspect, the present application provides a method for regulating the degree of pyroptosis or inflammatory necrosis of neuronal cells, comprising regulating the expression of lncMCL1 in the neuronal cells to regulate the degree of pyroptosis or inflammatory necrosis of the neuronal cells.

[0021] The regulation can be performed in vivo or in vitro.

[0022] In one embodiment, the regulation is to increase or decrease the degree of pyroptosis or inflammatory necrosis of the neuronal cells, depending on the purpose of application. Preferably, the regulation is to decrease the degree of pyroptosis or inflammatory necrosis of the neuronal cells. The neuronal cells are as described above in the first aspect.

[0023] Inflammatory necrosis and pyroptosis are two forms of cell death, both of which are involved in the neuroinflammatory response and can cause the release of inflammatory factors, activation of the inflammatory cascade, and neuronal damage and dysfunction. They induce and promote each other, forming a vicious cycle. Inflammatory necrosis of neurons triggers pyroptosis, and the inflammatory factors released by pyroptosis exacerbate neuroinflammation, leading to more neuronal death and further aggravating the condition of epilepsy. One common feature of the two forms is that they can activate inflammasomes, for example, cause an increase in the expression of NLRP3 inflammasome signaling pathway-related proteins and / or an increase in the content of molecular markers of inflammatory necrosis in the neuronal cells. In one embodiment, the NLRP3 inflammasome signaling pathway-related proteins can include at least one of NLRP3, GSDMD-FL, GSDMD-N, pro-Caspase1, and c-Caspase1; and the molecular markers of inflammatory necrosis can include at least one of TNFα, IL-1β, and IL-18.

[0024] In a preferred embodiment, the modulating comprises enhancing or reducing the expression of IncMCL1 in the neuronal cell. In a specific embodiment, the expression of IncMCL1 in the neuronal cell is enhanced by overexpression. In another specific embodiment, the expression of IncMCL1 in the neuronal cell 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 comprising a first strand having a nucleotide sequence of SEQ ID NO: 2 and a second strand having a nucleotide sequence of SEQ ID NO: 3, and the siRNA2 comprising a first strand having a nucleotide sequence of SEQ ID NO: 4 and a second strand having a nucleotide sequence of SEQ ID NO: 5.

[0025] In an embodiment, the modulating method comprises modulating (e.g., reducing) the degree of pyroptosis or inflammatory necrosis of the neuronal cell in vitro by modulating the expression of IncMCL1 in the neuronal cell using the substance for enhancing the expression of IncMCL1 in the neuronal cell according to the first aspect.

[0026] In a specific embodiment, the expression amount of NLRP3 inflammasome signaling pathway related protein and / or the content of molecular markers of inflammatory necrosis in the modulated cell is reduced by more than 30%, further by 35%, 40%, 45%, 50%, 60% and more, compared to before the expression of IncMCL1 is modulated. In a specific embodiment, the expression amount of NLRP3 inflammasome signaling pathway related protein and / or the content of molecular markers of inflammatory necrosis in the modulated cell is increased by more than 30%, further by 35%, 40%, 45%, 50%, 60% and more, compared to before the expression of IncMCL1 is modulated.

[0027] The method as described above is for a direct purpose other than disease diagnosis and / or disease treatment. The above-mentioned application or method is an application or method other than disease diagnosis. The above-mentioned application or method is not for the direct purpose of obtaining a disease diagnosis result or health condition of a living human or animal body.

[0028] In a third aspect, the present application provides a pharmaceutical composition comprising a substance for enhancing the expression of IncMCL1 in a neuronal cell and a pharmaceutically acceptable carrier.

[0029] The pharmaceutical composition as described above, the pharmaceutically acceptable carrier can be a substance for helping the substance to effectively enter cells and exert effects, which can be determined according to the purpose of treatment, the stability of the substance, the required targeting, the route of administration and other factors.

[0030] In the present application, the drug of the first aspect and the pharmaceutical composition of the third aspect can adopt various preparation forms according to the drug release mode, including but not limited to ordinary preparation, sustained-release preparation, controlled-release preparation and various microparticle drug delivery systems. In a specific embodiment, the preparation can be in the form of an injection.

[0031] In a fourth aspect, the present application provides a method for alleviating and / or treating a disease related to the loss of neuronal function, comprising administering to a subject a therapeutically effective amount of the substance for enhancing the expression of lncMCL1 in neuronal cells of the first aspect.

[0032] The subject in the method as described above refers to a subject suffering from a disease, disorder or dysfunction related to the loss of neuronal function, for example, a patient clinically diagnosed as having epilepsy, and further, the subject has temporal lobe epilepsy. The disease, disorder or dysfunction related to the loss of neuronal function can be status epilepticus caused by epilepsy. There can be an inflammatory response in the neurons of the subject with the disease, disorder or dysfunction related to the loss of neuronal function, especially an inflammatory response triggered by the classical (caspase-1 dependent) pathway.

[0033] The subject in the method as described above can be a mammal, which can be selected from the group consisting of bovids, equids, felids, canids, leporids, suids, camelids, rodents and primates, 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, chimpanzees) and humans, preferably rats, mice and humans.

[0034] The administration dose of the substance for enhancing the expression of lncMCL1 in neuronal cells in the method as described above is variable depending on the mode of administration, the route of administration, the age and / or weight of the individual, and the condition of the individual being treated, and is ultimately determined by the attending physician.

[0035] In a fifth aspect, the present application provides a method for screening a candidate drug capable of alleviating and / or reducing and / or treating epilepsy, comprising:

[0036] An in vitro neuronal injured cell model is established, and the test substance is contacted with the injured neuronal cells. After the contact, it is measured whether there is overexpression of lncMCL1 in the neuronal cells. If there is overexpression, the test substance is taken as a candidate drug.

[0037] In one embodiment, the disease, disorder or dysfunction associated with loss of neuronal function can be epilepsy or status epilepticus caused by epilepsy.

[0038] In one embodiment, the neuronal injury model can be a hippocampal neuronal injury model. In a specific embodiment, in the hippocampal neuronal injury model, the injured hippocampal neuronal cells undergo inflammatory necrosis and / or pyroptosis.

[0039] In the present application, the hippocampal neuronal injury model can employ a cell model or an animal model. The cell model can include, but is not limited to, a chemical injury model, e.g., a sodium arsenite injury model; an oxidative stress model, e.g., a hydrogen peroxide model; an inflammatory factor stimulation model, e.g., a lipopolysaccharide and cytokine combined stimulation model; a glutamate excitotoxicity model, e.g., a glutamate and its analogs injury model, etc. The neuronal cells used in the cell model can be a commonly used hippocampal neuronal cell line, e.g., mouse H-22 cell line. The animal model can include, but is not limited to, a traumatic brain injury model, e.g., a fluid percussion injury (FPI) model; an ischemia-hypoxia model, e.g., a bilateral common carotid artery ligation model; a chemical injury model, e.g., a sodium arsenite injury model and a PAPP inhibitor model; an inflammatory mediator injury model, e.g., an LPS-induced model, an experimental autoimmune encephalomyelitis model; a gene and protein level regulation model, e.g., a miRNA regulation model, a protein kinase modulator model, etc.

[0040] In a specific embodiment, the hippocampal neuronal injury model can employ a glutamate excitotoxicity cell model, i.e., glutamate and its analogs (e.g., kainic acid, NMDA) are used to treat hippocampal neuronal cells to induce excitotoxic injury of hippocampal neurons. This model is commonly used to study the mechanism of neuronal injury in neurological diseases such as epilepsy, cerebral hemorrhage, etc.

[0041] Therefore, in one embodiment, the screening method as described above comprises the following steps: establishing a glutamate excitotoxicity cell model (i.e., a hippocampal neuronal injury model), contacting a test substance with injured hippocampal neuronal cells in the presence of glutamate or its analogs, and measuring whether overexpression of lncMCL1 exists in the injured hippocampal neuronal cells after the contacting, and if overexpression exists, the test substance is considered as a candidate drug.

[0042] In one specific embodiment, the cells used in establishing the glutamate excitotoxicity cell model are hippocampal neuronal cell lines, preferably mouse H-22 cell lines. Methods for measuring lncMCL1 overexpression are well known in the art, for example, quantitative PCR (qRT-PCR), gene chips, Western blotting, immunohistochemical techniques, cell function experiments, etc.

[0043] The present application proves, through in vitro and in vivo experiments, that lncMCL1 can be used as a key regulator of pyroptosis or inflammatory necrosis of hippocampal neurons in epilepsy, can effectively regulate pyroptosis or inflammatory necrosis of neuronal cells, and can be used to relieve, alleviate or treat diseases related to the two mechanisms (for example, epilepsy or status epilepticus caused by epilepsy). BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Down-regulation of lncMCL1 expression in neurons in the temporal lobe tissue of patients with temporal lobe epilepsy; wherein A is the heat map analysis result of differential lncRNA and mRNA expression between the temporal lobe cortex (TLE) of patients with temporal lobe epilepsy and the temporal lobe cortex (Control) of patients with brain trauma; B is the mRNA data enrichment analysis comparison between the control and TLE cortex based on functional annotation; C is the Venn diagram of lncRNA conserved 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 epilepsy mice; E is the RT-qPCR analysis of lncMCL1 expression in the hippocampus of PTZ-induced epilepsy mice; F is the RT-qPCR analysis of lncMCL1 expression in the ipsilateral hippocampus of KA-induced epilepsy rats; G is the violin plot of lncMCL1 expression in the temporal lobe cortex of TLE patients and the temporal lobe cortex of patients with brain trauma; H is the correlation analysis of lncMCL1 expression in the hippocampus and TLE and history of epilepsy; I is the detection of lncMCL1 expression in the cytoplasm and nuclear fractions by RT-qPCR (n=3); J is the localization of lncMCL1 in the cortex samples of TLE patients detected by RNA FISH and immunofluorescence staining; K is the cell localization of lncMCL1 in the hippocampus of KA-induced epilepsy mice detected by RNA FISH and immunofluorescence staining of NeuN and GFAP; L is the localization and expression of lncMCL1 in the CA1, CA3 and DG regions of the ipsilateral and contralateral hippocampus of KA-induced epilepsy mice detected by RNA FISH, the left graph is a panoramic view of the hippocampus, and the right graph is a local view of the CA1, CA3 and DG regions.

[0045] Figure 2A is mRNA data enrichment analysis based on functional annotation comparing ov-NC and ov-lncMCL1 HT-22 cells; B is gene set enrichment analysis showing that high lncMCL1 expression is negatively correlated with inflammatory response in neurons; C is immunoblotting detection of NLRP3 inflammasome signaling (NLRP3, c-Caspase1, IL1β and IL18) in hippocampus of TLE patients and controls; D is quantification of C corresponding results; E is immunoblotting detection of NLRP3 inflammasome signaling (NLRP3, c-Caspase1, IL1β and IL18) in temporal cortex of TLE patients and controls; F is quantification of E corresponding results; G is immunofluorescence staining of NLRP3 and NeuN in hippocampus and temporal cortex of controls and TLE patients; H is immunofluorescence staining of c-Capase1 and NeuN in hippocampus and temporal cortex of controls and TLE patients; I is immunofluorescence staining of NLRP3 and NeuN in hippocampus of KA-induced epilepsy mice and TLE mice.

[0046] Figure 3 Overexpression of lncMCL1 in vitro inhibits glutamate-induced hippocampal neuronal pyroptosis; A is RT-qPCR analysis of lncMCL1 expression in HT-22 cells treated with different concentrations of glutamate (mean ± SEM, n=3, one-way ANOVA); B is IF staining of NLRP3 and c-Caspase1 in control and Glu-treated HT-22, Scarlbar is 50 μm; C is immunoblotting detection of NLRP3 inflammasome signaling (NLRP3, c-Caspase1, IL1β and IL18) in control and Glu-treated HT-22; D is quantification of C corresponding results, (mean ± SEM, n=4-5, Student's t-test); E is immunoblotting detection of cell pyroptosis markers in control and Glu-treated HT-22 transfected with ov-NC or ov-lncMCL1; F is quantification of E corresponding results, (mean ± SEM, n=2-3, two-way ANOVA); G is ELISA detection of IL18 / IL1β concentration 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 IF staining of NLRP3 in control and Glu-treated HT-22 transfected with ov-NC or ov-lncMCL1; I is IF staining of c-Caspase1 in control and Glu-treated HT-22 transfected with ov-NC or ov-lncMCL1.

[0047] Figure 4To knock down lncMCL1 in vitro to promote glutamate-induced pyroptosis of HT-22 cells; wherein, A is RT-qPCR analysis of lncMCL1 expression to assess siRNA interference efficiency, (mean ± SEM, n = 3, one-way ANOVA); B is immunoblotting to detect pyroptosis markers in control and Glu-treated HT-22 transfected with NC or siRNA; C is quantification of the results corresponding to B, (mean ± SEM, n = 3, two-way ANOVA); D is ELISA to detect the concentration of IL18 / IL1β in the medium of control and Glu-treated HT-22 transfected with NC or siRNA; E is IF staining of NLRP3 in control and Glu-treated HT-22 transfected with NC or siRNA; F is IF staining of c-Caspase1 in control and Glu-treated HT-22 (transfected with NC or siRNA).

[0048] Figure 5 To knock out lncMCL1 by CRISPR / Cas9 to promote glutamate-induced pyroptosis of HT-22 cells; wherein, A is a schematic diagram of knocking down lncMCL1 using the CRISPR / Cas9 system; B is sequencing results confirming that CRISPR / Cas9 inserted lncMCL1 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 quantification of the results corresponding to D, (mean ± SEM, n = 3, two-way ANOVA).

[0049] Figure 6 To verify in vivo that lncMCL1 inhibits pyroptosis of hippocampal neurons in epilepsy; wherein, A is a timeline of the experimental design; B is the number of SRS from the 28th to the 35th day after SE in epileptic mice with or without overexpression of lncMCL1 by video monitoring; C is a violin plot of the total seizure of KA-induced 7 days in the control and overexpression of lncMCL1 groups; D is an electroencephalogram (EEG) time-frequency plot showing the change in brain waves of PTZ-induced mice in the control and overexpression of lncMCL1 groups; E is a violin plot of the total seizure of PTZ-induced 7 days in the control and overexpression of lncMCL1 groups; F is Nissl staining to check the number of neurons in the ipsilateral and contralateral hippocampal CA1 and CA3 regions of TLE mice, the left panel is a panoramic view of the hippocampus, and the right panel is the CA1 and CA3 regions; G is quantification of the number of neurons, 3 positions were selected from each region for analysis, (mean ± SEM, n = 3, one-way ANOVA); H is immunofluorescence staining of NLRP3, c-Capase1, NeuN and GFAP in the hippocampus of control and TLE mice.

[0050] The above data are shown as mean ± SEM of independent experiments, *** indicates P<0.001, ** indicates P<0.01, * indicates P<0.05, ns indicates no significant difference. DETAILED DESCRIPTION

[0051] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. They should not be understood as limiting the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of the present application. In the description of the present application, it should be understood that the terms used are only for the purpose of describing, and should not be understood as indicating or implying relative importance.

[0052] In the following examples, the experimental methods are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0053] Male C57 BL / 6 mice (body weight 18 g-20 g) and Sprague-Dawley rats (body weight 200 g-220 g) used in the following examples were from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were raised in a constant temperature room, and were raised according to a standard 12-hour light / dark cycle, and could freely obtain standard rodent feed and water. All animal experimental procedures were approved by the Ethics Committee of Beijing Tiantan Hospital, Capital Medical University (Ethics Numbers AEEI-2018-200 and AEEI-2019-097).

[0054] Mouse neuronal cell lines HT-22 and HEK-293T cells (derived from ATCC (American Type Culture Collection, USA)) were cultured in Dulbecco's Modified Eagle Medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (all from Gibco, Grand Island, NY, USA) at 37°C and 5% CO2. For glutamate treatment, cells were incubated in DMEM medium containing 5 mM glutamate for 24 hours. For IL1β treatment, cells were incubated in DMEM medium containing 75 and 100 ng / ml IL1β for 6 hours and 24 hours, respectively. For IL18 treatment, cells were incubated in DMEM medium containing 75 and 100 ng / ml IL18 for 6 hours and 24 hours, respectively.

[0055] The nucleotide sequences involved in the following examples are shown in Table 1:

[0056]

[0057] Note: In Table 1, the bolded and underlined part of the sequence corresponding to SEQ ID NO:1 is the target sequence of sgRNA.

[0058] CRISPR-mediated lncMCL1 gene knockout in mouse HT-22 cell line

[0059] The target sequence for lncMCL1 sgRNA was designed by Hanheng Biotechnology (Shanghai) Co., Ltd. To prepare CRISPR lentivirus, HEK293T cells were seeded in 100 mm culture dishes and cultured using Lipofiter according to the manufacturer's instructions. TM The transfection system consisted of co-transfection with 10 μg lentiCRISPRv2-gRNA or lentiCRISPRv2 control plasmid (Addgene, MA, USA, plasmid #52961), 10 μg psPAX2 plasmid, and 5 μg PMD2G plasmid. Culture supernatant containing lentivirus was collected at 48 and 72 hours post-transfection and used to infect HT-22 cells for 16–24 hours. Polyclonal KO cell lines were obtained one week later, and subsequently screened in 96-well plates for 1–2 months to obtain monoclonal cell lines with stable target gene knockout.

[0060] RNA sequencing and data analysis

[0061] HT-22 cells transfected with 80 MOI LV-MCL1 virus were collected for RNA sequencing. RNA-seq was performed using a HiSeq3000 (Illumina) at Shanghai Biotechnology Co., Ltd. to detect the mRNA expression profile of HT-22 cells overexpressing lncMCL1. Transcriptional expression analysis of the RNA-seq experiments was performed using HISAT, and reference annotation was added to generate FPKM values ​​for known gene models. Differentially expressed genes were identified using edgeR. Fold change was also estimated based on FPKM in each sample. Differentially expressed genes were selected using the following filtering criteria: FDR ≤ 0.05 and fold change ≥ 2.

[0062] RNA extraction and RT-qPCR

[0063] Total RNA was extracted using TRIzol reagent (Invitrogen) and cDNA was acquired using MonScript™ RTII All-in-One Mix (Monad) according to the manufacturer’s instructions, unless otherwise stated. RT-qPCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme) and products were quantified using the CFX Connect Real-Time PCR Detection System (Bio-Rad). Relative gene expression levels were normalized to the level of GAPDH and 2 -ΔΔCt Methodology calculations.

[0064] Immunofluorescence (IF) staining

[0065] Cells plated on coverslips or frozen mouse brain sections were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 5 min, and blocked with QuickBlock™ immunostaining blocking buffer (Beyotime) for 1 h at room temperature. Then, cells or brain tissues were incubated with the following primary antibodies overnight at 4°C: anti-NLRP3 (Abeam, ab214185, 1:100), anti-Cleaved-Caspase-1 (Cell Signaling Technology, 89332, 1:100), anti-NeuN (Abeam, ab1024224, 1:300), anti-GFAP (Abeam, ab53554, 1:500). The next day, cells or brain tissues were incubated with the corresponding secondary fluorescent antibodies (Proteintech, SA00013-2, SA00013-3 and SA00013-4).

[0066] Western blot analysis

[0067] Each group of brain tissue or cells was homogenized on ice with RIPA buffer (Bi Yun Tian Biotechnology, China) plus 1 : 1000 protease inhibitor cocktail and 1 : 1000 phosphatase inhibitor cocktail (MedChemExpress). After quantification with a BCA kit (Thermo Fisher Scientific), equal amounts of cell lysate or brain tissue lysate proteins were boiled, denatured, separated by SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane with SDS-PAGE sample loading buffer (Bi Yun Tian). Then, the membrane was blocked with 5% skim milk dissolved in TBST and incubated with primary antibodies overnight, then with secondary antibodies and exposed to Immobilon Crescendo Western HRP substrate (Millipore). The primary antibodies used were as follows: anti-NLRP3 (Abeam, ab210491, 1 :800), anti-pro-Caspase 1 (Abeam, 179515 and ab1872, 1 :500), anti-Cleaved Caspase-1 (Cell Signaling Technology, 89332, 1 : 1000), anti-IL1 b (Abeam, ab9722, 1 :500), anti-IL18 (Proteintech, 10633-1-AP, 1 :500), anti-GSDMD (Abeam, ab209845, 1 :500), anti-GSDMD-N (CST, 50928, 1 : 1000), anti-ACS (Cell Signaling Technology, 67873, 1 :500), anti-TNFa (Abeam, ab6671, 1 :500), anti-GAPDH (Abeam, ab181602, 1 :3000). Secondary antibodies were anti-rabbit IgG (Cell Signaling Technology, 7074, 1 :2000) and anti-mouse IgG (Cell Signaling Technology, 7076, 1 :2000).

[0068] fluorescence in situ hybridization

[0069] Paraffin-embedded sections (4 pm thickness) were deparaffmized, dehydrated and treated with 1 M sodium thiocyanate. Sections were then digested in a pepsin solution, fixed in 4% formaldehyde, dehydrated by sequential immersion in 70%, 85% and 100% ethanol, and air-dried. Sections were incubated with digoxigenin (DIG)-labelled probes (probes 1-3 shown in Table 1) followed by incubation with DyLight 594-conjugated IgG grade fraction (Abcam, ab96873) conjugated to monoclonal mouse anti-DIG antibody (Abcam, ab116590). Nuclei were counterstained with DAPI. Sections were scanned and analysed as described above.

[0070] Immunoprecipitation (IP)

[0071] IP assays were performed using 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. Cell lysates were combined with IP antibody to make 500 pL antibody / lysate solution and the solution was incubated at 4°C overnight. Then, the solution was incubated with 25 pL Pierce Protein A / G magnetic beads for 1 hour at room temperature. Collected bead-protein complexes were heated to 96-100°C for 10 minutes in LaneMarker sample buffer (Thermo Fisher Scientific) before SDS-PAGE.

[0072] RNA pull-down and mass spectrometry analysis

[0073] RNA pull-down was performed using Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) following the manufacturer’s protocol. Biotin RNA labelling mix and T7 RNA polymerase (Invitrogen) were used to transcribe mouse IncMCL1 sequence in vitro. Biotinylated IncMCL1 RNA was then incubated with streptavidin-conjugated magnetic beads and total lysate of HT-22 cells for 2 hours at room temperature. After incubation, RNA-protein complexes bound to beads were washed four times with wash buffer. Proteins were then eluted and resuspended in protein lysis buffer. Finally, eluted proteins were analysed by SDS-PAGE and subjected to mass spectrometry analysis.

[0074] RNA immunoprecipitation

[0075] RNA immunoprecipitation (RIP) assay was performed using RNA immunoprecipitation (RIP) kit (BersinBio) according to the manufacturer’s instruction. Briefly, 2 x 107HT-22 cells were harvested and lysed with polysome lysis buffer. Cell extracts were co-immunoprecipitated with anti-DDX3X antibody (Santacruz, sc-365768) or anti-NEDD4 (Proteintech, 83112-1-RR), and the recovered RNA was subjected to qRT-PCR analysis using lncMCL1 primer pair. Total RNA (input control) and normal mouse IgG control were also determined to confirm that the detected signal was from the RNA specifically bound to DDX3X or NEDD4.

[0076] Nissl staining

[0077] Mice were perfused with 0.01 M phosphate-buffered saline (PBS; pH 7.4) and then with 4% paraformaldehyde in 0.01 M PBS. The brain was fixed with 4% paraformaldehyde in 0.01 M PBS at 4 °C overnight and then put into a 30% sucrose solution until the brain sank to the bottom of the cavity. The OCT-embedded tissue was cut into 25 cm thin sections and mounted on pre-coated glass slides (Golden Bridge, Shanghai, China). The slides were stained with cresyl violet (Beyotime Institute of Biotechnology, Shanghai, China) for 10 min. Then the slides were rinsed with distilled water, dehydrated with different concentrations of ethanol (70%, 80%, 90%, 100%), cleaned with xylene, and covered with neutral resin. Neurons in the hippocampal CA3 and CA1 regions were analyzed. The average number of neurons in three 40x light fields at the same section position of each mouse was calculated.

[0078] Statistical analysis

[0079] 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 from two different groups. Statistical comparisons of multiple groups were analyzed using one-way or two-way analysis of variance (ANOVA), followed by appropriate post-hoc tests. Data are presented as mean ± standard error of the mean (SEM). P < 0.05 was considered significant.

[0080] Example 1, Screening of TLE patient temporal lobe cortex lncRNA expression profile

[0081] Temporal lobe tissue from patients with temporal lobe epilepsy (TLE) was collected for microarray analysis, and temporal lobe cortex from patients who underwent surgery after brain trauma was used as a control group (n = 4). RNA samples were first reverse-transcribed into cDNA, and then the cDNA samples were labeled and hybridized to the array according to the Agilent Technologies single-color microarray gene expression analysis protocol (Agilent Technologies, Santa Clara, CA, USA). When the fold change (FC) was ≥ 2 and P < 0.05, the lncRNA and mRNA were considered to be differentially expressed between the two groups of samples. The results of the analysis are shown in Figure 1 As shown in FIG. 1A, 490 differentially expressed mRNAs were found between the TLE patient group and the control group (Control), 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.

[0082] The differentially expressed genes were placed in the Database for Annotation, Visualization and Integrated Discovery (DAVID; http: / / david.abcc.ncifcrf.gov / ) to identify the molecular functions represented in the gene profiles using GO. As shown in FIG. 1B, 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, and positive regulation of acute inflammatory response, suggesting that neuroinflammation is one of the important pathological processes in the occurrence of epilepsy. Figure 1

[0083] Because lncRNAs are different from coding genes, miRNAs and circRNAs, they lack evolutionary conservation, whereas highly conserved genes often play an important role in maintaining the normal biological functions of an organism. Therefore, homology analysis was performed on the 736 differentially expressed lncRNAs (e-value less than le-5) using Blastn homology analysis to identify lncRNAs conserved between species. The results are shown in FIG. 1C, which shows that 140 lncRNAs are conserved between humans and mice (Mus musculus), 63 lncRNAs are conserved between humans and rats (Rat), and 5 lncRNAs are conserved between humans, rats and mice, which are lnc-MCL1-2:1 (NONHSAT006310.2, abbreviated as lncMCL1), NR_002139, ENST00000501122, ENST00000562284 and ENST00000605417, respectively. Figure 1

[0084] 2. lncMCL1 is down-regulated in the occurrence of epilepsy

[0085] ​​2.1, Construction of KA-induced epilepsy model in the amygdala: 0.3 μL of KA (1 μg / μL, Sigma-Aldrich, St. Louis, Missouri, USA) was injected into the right amygdala of mice (0.94 mm behind the bregma, 2.75 mm lateral, 4.75 mm deep) and 0.7 μL of KA was injected into the right amygdala of rats (2.76 mm behind the bregma, 4.5 mm lateral, 8.6 mm deep). The sham control group received the same volume of intracerebral injection of normal saline. The mice were implanted with cortical electroencephalogram electrodes, and the spontaneous recurrent seizures (SRS) of the epileptic mice were continuously video-EEG monitored (24 hours / day). The SRS was scored according to the Racine scale. Racine scale: 0, no seizures; I, facial myoclonus, piloerection, and scratching and climbing; II, I plus head nodding; III, II plus forelimb clonus; IV, III plus hindlimb clonus or tetany; V, IV plus continuous rearing and rolling. Only the SRS frequency of the IV and V stage animals was recorded and analyzed. The video-EEG monitoring of any level of seizures above indicated the success of modeling. Then, the expression pattern of lncMCL1 in the hippocampus of mice and rats was analyzed at 1, 10, and 30 days after KA injection. The analysis results are shown in Figs. 1D and 1F, respectively. Figure 1 As can be seen from Figs. 1D and 1F, the expression of lncMCL1 was down-regulated in the process of epilepsy in the mouse (Mouse) and rat (Rat) epilepsy models.

[0086] 2.2, Construction of PTZ model: The mice were implanted with cortical electroencephalogram electrodes, and 35 mg / kg of PTZ was injected intraperitoneally every other day. The 30-minute video-EEG monitoring of the epileptic behavior was performed after each injection of PTZ. The seizure stage was scored as follows: 0, normal behavior, no abnormalities. 1, immobility, prone position. 2, head nodding, facial, forelimb, or hindlimb myoclonus. 3, continuous generalized myoclonus, clonic convulsions, tail rigidly raised. 4, upright, tonic seizures, lateral fall. 5, tonic-clonic seizures, prone fall, running and jumping. 6, death. In order to produce fully kindled animals, once the animal experienced a seizure with a score of 5 (tonic-clonic seizures), the injection was completed within the next three administrations. In order to evaluate the vulnerability of the mice injected with AAV to PTZ, 35 mg / kg was injected every other day for a total of 6 times for each mouse. The video-EEG monitoring of any level of seizures above indicated the success of modeling. The expression pattern of lncMCL1 in the hippocampus of mice was analyzed after PTZ injection. The analysis results are shown in Figs. 2D and 2F, respectively. Figure 1As shown in FIG. 2E, the expression of LncMCL1 in the hippocampus was also significantly reduced in a mouse epilepsy model induced by pentyltetrazole (PTZ).

[0087] 2.3, the analysis of lncMCL1 expression was expanded to 20 TLE patient samples, and the analysis results are shown in FIG. 2C. Figure 1 As shown in FIG. 2G, it can be seen that the expression of lncMCL1 in TLE patients is significantly reduced.

[0088] 2.4, the correlation between the expression of lncMCL1 in the hippocampus of TLE patients and the history of epilepsy of the patients was analyzed, and the results are shown in FIG. 2H. Figure 1 As shown in FIG. 2H, it can be seen that the expression of lncMCL1 in the hippocampus of TLE patients is negatively correlated with the history of epilepsy.

[0089] In summary, these results show that the conserved lncMCL1 is reduced in epilepsy and may play an important role in the occurrence of epilepsy.

[0090] 2.5, in order to further determine the subcellular localization of lncMCL1, the cytoplasm and nuclear RNA of HT-22 cells were separated and purified according to the manufacturer's instructions using NE-PER nuclear and cytoplasmic extraction reagent (#78833, Pierce, Rockford, IL), and then qRT-PCR was performed. The results are shown in FIG. 2I. Figure 1 As shown in FIG. 2I, it is shown that lncMCL1 is mainly located in the cytoplasm.

[0091] 2.6, paraffin-embedded sections of TLE patient cortex samples and hippocampal tissues of KA-induced mouse and rat epilepsy models were prepared and fluorescence in situ hybridization (FISH) analysis was performed; specifically including: the paraffin-embedded sections (4 microns thick) were deparaffinized, dehydrated and treated with 1 M sodium thiocyanate. Then the sections were digested in pepsin solution, fixed in 4% formaldehyde, dehydrated by immersion in 70%, 85% and 100% ethanol, and then air dried. The sections were incubated with digoxin (DIG) labeled probes 1-3, and then incubated with DyLight 594 conjugated IgG grade (Abeam, ab96873) conjugated with monoclonal mouse anti-DIG antibody (Abeam, ab116590). The nuclei were counterstained with DAPI. The results are shown in FIGS. 2J-2K. Figure 1 As shown in FIGS. 2J-2K, it is indeed revealed that lncMCL1 is distributed in the cytoplasm, and the expression of lncMCL1 in the ipsilateral and contralateral hippocampus of KA-injected epilepsy mice is reduced. Double staining of lncMCL1 and the astrocyte protein marker GFAP in brain samples of epilepsy mouse and rat models shows that lncMCL1 is mainly located in the cytoplasm of neurons, not in astrocytes.

[0092] 2.7, RNA FISH detection of IncMCL1 in ipsilateral and contralateral hippocampal CA1, CA3 and DG regions of KA-induced epileptic mice, results as shown in Figure 1 Fig. 2K, L, the expression of IncMCL1 was decreased in ipsilateral and contralateral hippocampus of KA-injected epileptic mice (p < 0.05, t-test). Figure 1 Fig. 2K, L). Double staining of IncMCL1 and astrocyte protein marker GFAP in brain samples of epileptic mice and rat models showed that IncMCL1 was mainly located in the cytoplasm of neurons, not in astrocytes (Fig. 2K, L). Figure 1 Fig. 2K, L).

[0093] 3, NLRP3 inflammasome-mediated pyroptosis activation in neurons of epileptic tissues.

[0094] To explore the biological function of IncMCL1, IncMCL1 was overexpressed in HT-22 cells, and then the transcriptome RNA was screened using RNA-Seq. After overexpression of IncMCL1 in HT-22 cells, 103 RNAs were up-regulated and 246 RNAs were down-regulated (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 response (GO terms: cellular response to interleukin-1, chemokine activity, chemokine receptor binding), indicating that IncMCL1 was related to neuronal inflammation (Fig. 3A-B). Figure 2 A-B).

[0095] Pyroptosis is a kind of 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-1 beta (IL-1 beta) and interleukin-18 (IL-18). Given that pyroptosis occurs in neurons in epilepsy and other nervous system diseases, we found that the biological function of IncMCL1 in neurons was related to the above interleukin-1, so we speculated that IncMCL might be related to pyroptosis.

[0096] Then, NLRP3 inflammasome-mediated neuronal pyroptosis proteins, including NLRP3, Caspase1, IL1b and IL18, were detected in temporal lobe epilepsy. Western blot analysis showed that the expression of NLRP3, Caspase1, IL1b and IL18 in the hippocampus of TLE was significantly increased compared with the control group (Fig. 4A-D). Figure 2C, D) and the protein levels of NLRP3, cleaved-Caspase1 (c-Caspase1), IL1β and IL18 were significantly increased in the temporal lobe cortex of TLE patients (E, F). Figure 2 E, F). Double immunofluorescence staining was used to colocalize NLRP3 and c-Caspase1 with the neuronal marker NeuN and the astrocyte marker GFAP, and the results further showed that NLRP3 and c-Caspase1 were mainly located in neurons and astrocytes in the hippocampus and temporal lobe cortex of TLE patients (G, H) and in the hippocampus of KA-induced TLE mouse models at different stages of epileptogenesis (I), indicating that NLRP3 inflammasome was activated in TLE. Figure 2 G, H), and the hippocampus of KA-induced TLE mouse models at different stages of epileptogenesis (I), indicating that NLRP3 inflammasome was activated in TLE. Figure 2 I).

[0097] Example 2, in vitro experiments confirmed that lncMCL1 inhibited pyroptosis of epileptic hippocampal neurons

[0098] This example discusses the role of lncMCL1 in glutamate-induced hippocampal neuron injury and its relationship with pyroptosis triggered by status epilepticus (SE).

[0099] 1. A glutamate-treated HT22 (mouse hippocampal neuron cell line) was used to establish an in vitro injured neuron model; 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, and the results are shown in Figure 3 A, which is consistent with the results of in vivo experiments, and the expression level of lncMCL1 in the cell line was significantly down-regulated by excessive glutamate. Western blot and immunofluorescence (IF) experiments were used to detect the protein expression level and mRNA expression level of NLRP3, GSDMD-FL, GSDMD-N, pro-Caspase1 and c-Caspase1 in the HT22 mouse hippocampal neuron cell line, and the results are shown in Figure 3 B-D, it can be seen that the expression levels of NLRP3, GSDMD-FL, GSDMD-N, pro-Caspase1 and c-Caspase1 in the cells were significantly increased after glutamate treatment compared with the control group, indicating that glutamate can induce inflammatory necrosis of neurons.

[0100] 2. The coding sequence of lncMCL1 (SEQ ID NO:1) was inserted into the lentiviral vector (pHBLV-CMV-MCS-EF1-Zsgreen1-T2A-puro). A GV358 viral vector carrying a scrambled sequence was used as a negative control (NC).

[0101] HT-22 cells were loaded at 3 × 10 5 Cells were seeded at a density of 30%-50% in 6-well plates. Transfection was performed at 80 MOI, with 1 ml of complete culture medium containing 40 μl Hitrans GP infection reagent, 20 μl NC lentiviral vector, and 20 μl LV-MCL1. 12-16 h post-infection, the medium containing the lentiviral vector was replaced with medium containing fresh serum. Two days after infection, cells were selected using 3 μg / ml puromycin. After selection, cells were treated with PBS buffer and 5 mM glutamate. Twenty-four hours after treatment, the expression of proteins related to the NLRP3 inflammasome signaling pathway, including NLRP3, ASC, c-Caspase1, GSDMD-N, TNFα, IL-1β, and IL-18, was analyzed using Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF). The results are shown below. Figure 3 As shown in EI. The results indicate that under glutamate treatment, overexpression of lncMCL1 significantly inhibited the expression of proteins related to the NLRP3 inflammasome signaling pathway, while no significant changes were observed under normal conditions.

[0102] 3. To verify the mechanism of action of lncMCL1, small interfering RNA (siRNA) technology was used to knock down lncMCL1. First, small interfering RNAs (siRNA1-3) targeting mouse lncMCL1 and a disordered control siRNA were synthesized. HT-22 cells were transfected with 20 nmol of lncMCL1 siRNA or disordered siRNA using Lipofectamine RNAiMAX (Invitrogen). After 8 hours of transfection in antibiotic-free medium, the cells were replaced with normal medium. Cells were collected for analysis 48 hours after transfection. The results are as follows: Figure 4 As shown in Figure A, siRNA1 and siRNA2 (but not siRNA3) significantly reduced the expression level of lncMCL1. Cells with reduced lncMCL1 expression were analyzed using the same analytical methods as in the overexpression experiment, and the results are as follows. Figure 4As shown in the BE diagram, knocking down lncMCL1 with 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.

[0103] 4. For example Figure 5 As shown in Figure AB, a lncMCL1 knockout monoclonal cell line was constructed using CRISPR-Cas9 gene editing technology. Simultaneously, the lncMCL1 knockout monoclonal cell line was treated with a 5 mM glutamate solution, with PBS buffer as a control. After 24 h of treatment, Western blot analysis was performed on proteins related to pyroptosis in the lncMCL1 knockout monoclonal cell line. The results are shown below. Figure 5 As shown in the DE diagram, it can be seen that Cas9-mediated lncMCL1 knockout and siRNA-mediated lncMCL1 knockdown are consistent at the molecular level.

[0104] Example 3: In vivo experiments confirmed that lncMCL1 inhibits pyroptosis in epileptic hippocampal neurons.

[0105] 1. The in vivo biological functions of lncMCL1 were investigated by overexpressing it using an adeno-associated virus (AAV) delivery system. For example... Figure 6 As shown in Figure A, the cDNA sequence of lncMCL1 (ENSMUST00000175032, SEQ ID NO:1) was cloned into the pHBAAV-CMV-MCS-EF1-mcherry AAV expression vector from Shanghai Hanheng Biotechnology Co., Ltd., to construct a CMV promoter-driven recombinant AAV carrying lncMCL1 (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 from Shanghai Jikai Gene Technology Co., Ltd., to construct a CMV promoter-driven recombinant AAV carrying DDX3X (AAV-DDX3X). These AAV vectors were diluted to a titer of 5 × 10⁻⁶. 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 the anterior fontanelle, 2.1 mm lateral, 1.9 mm depth). Two weeks after AAV injection, mice were given KA-induced epilepsy, and the frequency of chronic seizures in mice with and without lncMCL1 overexpression was monitored by video at weeks 5-6 following KA-induced epilepsy. Results are as follows. Figure 6 As shown in the BE study, compared with sham-operated mice, mice overexpressing lncMCL1 had a significantly lower frequency of recurrent epileptic seizures.

[0106] 2. The hippocampal tissues of overexpressing lncMCL1 mice (ov-lncMCL1) and its control mice (ov-NC) were subjected to Nissl staining, and the results are shown in FIGS. 1A-1F. Figure 6 ov-lncMCL1 pretreatment can protect SE-induced hippocampal neuron damage in the ipsilateral CA3, CA1 regions, and the contralateral CA1 and CA3 regions.

[0107] IF analysis showed that, as shown in FIG. 1H, ov-lncMCL1 pretreatment reduced the protein levels of NLRP3, c-Caspase1 and GSDMD-N. Figure 6

[0108] In summary, the above in vitro and in vivo experimental results show that, during the occurrence of epilepsy, the decrease of lncMCL1 loses the inhibitory effect on NLRP3 inflammasome, leading to pyroptosis of nerve cells and seizures.

[0109] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.​

Claims

1. The use of substances that enhance the expression of lncMCL1 in neuronal cells in the preparation of drugs for alleviating and / or treating diseases, symptoms or functional impairments related to neuronal function loss, wherein lncMCL1 is a lncRNA encoded by DNA as shown in SEQ ID NO:1; Substances that enhance lncMCL1 expression in neuronal cells are selected from at least one of A1-A3: A1) The nucleic acid molecule encoding the lncMCL1; A2) An expression cassette containing the nucleic acid molecules 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); The disease, condition, or dysfunction related to the loss of neuronal function is epilepsy or status epilepticus caused by epilepsy.

2. The application according to claim 1, characterized in that, The neurons are hippocampal neurons.

3. The application according to claim 1, characterized in that, The neurons are derived from the hippocampus or the cerebral cortex.

4. A pharmaceutical composition, characterized in that, This includes substances that enhance the expression of lncMCL1 in neuronal cells and pharmaceutically acceptable carriers; lncMCL1 is a lncRNA encoded by the DNA shown in SEQ ID NO:1; Substances that enhance lncMCL1 expression in neuronal cells are selected from at least one of A1-A3: A1) The nucleic acid molecule encoding the lncMCL1; A2) An expression cassette containing the nucleic acid molecules 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).

5. A method for screening candidate drugs that can alleviate and / or reduce and / or treat diseases, conditions, or functional impairments related to neuronal function loss, characterized in that, include: An in vitro neuronal injury cell model was established, and the test substance was brought into contact with the damaged neuronal cells. After contact, the presence of lncMCL1 overexpression in the damaged neuronal cells was measured. If overexpression was present, the test substance was selected as a candidate drug. lncMCL1 is a lncRNA encoded by the DNA shown in SEQ ID NO:1; The disease, condition, or dysfunction related to the loss of neuronal function is epilepsy or status epilepticus caused by epilepsy.

6. The method according to claim 5, characterized in that, The damaged neurons undergo inflammatory necrosis and / or pyroptosis.

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