Application of NORAD in regulation of hypoxic ischemic neuron death mode

Abstract

The invention discloses an application of NORAD in regulation and control of a hypoxic ischemic neuron death mode, and relates to the technical field of biomedicine. Experiments prove that the neuron overexpresses NORAD, so that the cell viability after hypoxia and ischemia is increased, and the mitochondrial DNA copy number is increased, that is, the neuron mitochondrial activity and metabolism can be improved through NORAD overexpression, the neuron cell viability after hypoxia and ischemia is increased, and the method can reduce neuron apoptosis and necrotic apoptosis increase after hypoxia and ischemia.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of NORAD-overexpressing neurons in altering cell death patterns during hypoxia and ischemia. Background Technology

[0002] Hypoxic-ischemic encephalopathy (HIE) is a serious perinatal complication that can lead to irreversible neurological damage, including cerebral palsy, cognitive impairment, and even death. Currently, hypothermia is the only clinically recognized neuroprotective measure, but its efficacy is limited. Therefore, exploring new therapeutic targets is crucial. Under hypoxic-ischemic conditions, neurons undergo apoptosis, necrosis, or autophagy—pyroptosis—through mechanisms such as impaired energy metabolism, oxidative stress, calcium overload, and opening of the mitochondrial permeability transition pore (mPTP). The degree of hypoxic-ischemic damage may determine the activated cell death pathways, which often occur in combination with several different death mechanisms. This makes research targeting a single pathway less effective, prompting the development of new therapeutic methods that can affect multiple targets and prevent neuronal death.

[0003] Long non-coding RNAs (lncRNAs) are transcripts exceeding 200 nucleotides. In hypoxic-ischemic diseases, the expression profile of lncRNAs is significantly altered, and they play crucial roles. Recent research on the role of lncRNAs in different pathological mechanisms of cerebral ischemia has provided new directions for research on the pathogenesis and targeted therapy of HIE. NORAD (Non-coding RNA Activated by DNA Damage) is a long non-coding RNA that plays important functions in various biological processes, particularly in maintaining genome stability, responding to DNA damage, and regulating the cell cycle. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method that can alter the mode of neuronal death in neonatal hypoxic-ischemic encephalopathy.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] On the one hand, the present invention provides the application of NORAD in the preparation of drugs for the prevention or treatment of hypoxic-ischemic encephalopathy.

[0007] Preferably, the drug is a drug that promotes NORAD expression.

[0008] Preferably, the drug that promotes NORAD expression contains a lentiviral overexpression vector for NORAD.

[0009] Preferably, the drug is a drug that promotes the vitality of oxygen-glucose deprivation OGD neurons and / or inhibits the death of oxygen-glucose deprivation OGD neurons.

[0010] Preferably, the drug upregulates the levels of Ripk3 and MLKL in oxygen-glucose deprived OGD neurons.

[0011] On the other hand, the present invention also provides a medicament for the prevention or treatment of hypoxic-ischemic encephalopathy, comprising a promoter that promotes NORAD expression.

[0012] Preferably, the drug is a drug that promotes the vitality of oxygen-glucose deprivation OGD neurons and / or inhibits the death of oxygen-glucose deprivation OGD neurons.

[0013] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0014] Preferably, the dosage form of the drug includes capsules, tablets, granules, oral preparations, emulsions, sprays, ointments, or suspensions.

[0015] On the other hand, the present invention also provides a neuronal cell overexpressing NORAD, wherein a lentivirus containing NORAD is transfected into a cultured neuronal cell.

[0016] Compared with existing technologies, the beneficial effects of this solution are:

[0017] This invention demonstrates through experiments that overexpression of NORAD in neurons increases cell viability by approximately 50% and increases mitochondrial DNA copy number after hypoxia and ischemia. In other words, NORAD overexpression can improve neuronal mitochondrial activity and metabolism, thereby increasing the activity of hypoxic and ischemic neurons. Furthermore, this method can reduce neuronal apoptosis after hypoxia and ischemia and increase necrotizing apoptosis. Attached Figure Description

[0018] Figure 1 The statistical results of the relative content of NORAD in the right brain tissue of mice 24 hours after HIBD by RT-PCR;

[0019] Figure 2 The results of FISH analysis of NORAD localization in P7 mouse brain tissue;

[0020] Figure 3 The statistical results of the relative NORAD content detected by RT-PCR at 1, 6, 12, 24 and 48 h after OGD in primary neurons;

[0021] Figure 4To detect the sublocalization of NORAD in primary neurons before and 24 hours after OGD using FISH (A: RT-PCR detection of NORAD distribution in the nucleus and cytoplasm after RNA extraction from the nucleus and cytoplasm; NEAT1 is a positive control for nuclear RNA, and GAPDH is a positive control for cytoplasmic RNA; B: FISH detection of 18S and NORAD in the cytoplasm of the positive control cells, scale bar: 5 μm; C: Colocalization detection of NORAD (red) with lysosomal marker Lamp1 (green) and mitochondrial marker Tom20 (green), scale bar: 10 μm);

[0022] Figure 5 Transmission electron micrographs of brain tissue from P8 wild-type and NORAD knockout mice (red arrows in A show apoptotic neurons, scale bar 2 μm; yellow arrows in B show mitochondrial swelling and mitochondrial cristae vacuoles, scale bar 500 nm).

[0023] Figure 6 The results of apoptosis and necrotizing apoptosis in brain tissue of P8 wild-type and NORAD knockout mice are shown in the figures. (A shows the Western blot analysis of p-Ripk1, MLKL, p-MLKL, Ripk3, and p-Ripk3, and the statistical analysis of p-MLKL and p-Ripk3; B shows the Western blot analysis of Cleaved caspase3, Bcl2, and Bax, and the statistical analysis of Cleaved caspase3, Bax / Bcl2; C shows the TUNEL staining image of the hippocampus in paraffin sections of brain tissue, with a scale bar of 500 μm on the left and 100 μm on the right; D shows the p-MLKL (green) and NeuN (red) staining image of the hippocampus in paraffin sections of brain tissue, with a scale bar of 500 μm on the left and 100 μm on the right; the section thickness is 5 μm.)

[0024] Figure 7 Statistical results of the relative contents of NORAD, Ripk1, Ripk3 and MLKL in wild-type and NORAD knockout mice 24 h after HIBD detection by RT-PCR;

[0025] Figure 8 Laser speckle imaging results of the right hemisphere of wild-type and NORAD knockout mice 24 hours after HIBD (A is a pseudo-color image, the selected area is the statistical region, and B is the statistical result of the average cerebral blood flow in the selected area).

[0026] Figure 9 Statistical results of the relative mtDNA content of wild-type and NORAD-overexpressing neurons after OGD detection by RT-PCR;

[0027] Figure 10Figure 1 shows the results of apoptosis and necrotizing apoptosis in wild-type and NORAD-overexpressing neurons after OGD (A is a statistical graph of cell viability measured by CCK8; B is a WB image of Bcl2 and Bax and statistical analysis of Bax / Bcl2; C is a statistical graph of the relative content of Ripk1, MLKL, and Ripk3 mRNA detected by RT-PCR at 12, 24, and 48 h after neuronal OGD; D is a WB image of Ripk1, MLKL, Ripk3, and p-MLKL and statistical analysis; Ctrl: control group, NORADLV: NORAD overexpression group, Ctrl OGD: control lentivirus oxygen-glucose deprivation group, NORADLV OGD: NORAD overexpression oxygen-glucose deprivation group). Detailed Implementation

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] The experimental materials and detection methods for the research indicators used in the following embodiments are as follows:

[0032] 1.1 Laboratory Animals

[0033] The HIBD model was constructed using postnatal day 7 (P7) SPF-grade C57BL / 6 mice (wild-type mice) and the primary neuron extraction was performed using gestational day 17 SPF-grade C57BL / 6 female mice (wild-type female mice), both purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. NORAD knockout mice were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.

[0034] 1.2 Construction of the HIBD Model

[0035] Wild-type mice and NORAD knockout mice were anesthetized with isoflurane in an induction anesthesia chamber, then fixed in a supine position on a 37°C heating pad and continuously anesthetized with isoflurane. The right common carotid artery was bluntly dissected, transected, and the incision was sutured and disinfected. The sutured mice were then fed and recovered with their mothers for 40 minutes. A hypoxic chamber was set at 35°C, with an oxygen concentration of 10% and a carbon dioxide alarm set for exceeding 5%. The mice were placed in the hypoxic chamber for 80 minutes to induce hypoxia, then removed and fed with their mothers. Wild-type HIBD mice and NORAD knockout HIBD mice were obtained.

[0036] 2.2 Primary neuron culture in mice

[0037] 1) Anatomy of fetal rat cerebral cortex and hippocampus

[0038] Pregnant mice were euthanized with isoflurane after induction of anesthesia, then placed in a supine position via cervical dislocation. After removing the placenta, the placental tissue was dissected to extract the fetal mice. The fetal mice were briefly sterilized by immersing them in a 10cm culture dish containing 75% alcohol before being transferred to a biosafety cabinet. The heads were severed with scissors and placed in a 10cm culture dish containing 15mL of pre-chilled PBS. The skull was carefully removed with forceps, and the brain tissue was placed in another culture dish containing pre-chilled PBS. The meninges were then carefully removed in PBS, and the cerebellum, midbrain, and hypothalamus were excised, leaving only the cortex and hippocampus.

[0039] 2) Digestion of fetal rat cerebral cortex and hippocampus

[0040] The cerebral cortex and hippocampal tissue obtained from the dissection of fetal mice were placed in a 60 mm culture dish containing 5 mL of pre-chilled digestive solution. After separating the brains of 10 mice, the tissue blocks were cut into pieces of approximately 3 mm using forceps and scissors. 3 Add 100 μL of DNase working solution to the small fragments, gently shake to mix, and place in a CO2 cell culture incubator for 30 min. Gently shake the culture dish every 10 min during this period. Place a 70-mesh cell sieve onto a 50 mL centrifuge tube beforehand. After 30 min, remove the culture dish from the CO2 cell culture incubator, add 5 mL of neutralization solution, gently shake to mix, and stop the digestion.

[0041] 3) Culture of primary neurons

[0042] Gently pipette the culture medium containing tissue 10 times with a 1mL pipette tip. Tilt the culture dish at 30°C and let it stand for 2 minutes until the tissue fragments settle. Transfer 3mL of cell supernatant onto a cell strainer to filter out tissue fragments. Repeat the process of gently pipetting the remaining tissue fluid 10 times with a 1mL pipette tip, letting it stand, and transferring the supernatant onto a cell strainer until the fragments in the culture dish cannot be digested into a cell suspension. Centrifuge the filtered cell suspension at 200g for 5 minutes, discard the supernatant, and add neuronal culture medium. Prepare 6-well plates at 3 × 10⁻⁶ cells / well.6 1 cell / 2 mL culture medium, 1.5 × 10⁶ cells / well, 12-well plate 6 10 cells / 1 mL culture medium, 24-well plate 7 × 10⁶ cells / mL culture medium 5 Inoculate 1 cell per 0.5 mL culture medium plate, mix the cells using the cross-linking method, and incubate in a CO2 cell culture incubator. After 1 day, completely replace the medium with neuronal culture medium, and after 3 days, replace half the medium. Culture for 6-7 days until ready for use.

[0043] 2.3 Primary mouse neuron OGD experiment

[0044] Pre-hypoxic DMEM sugar-free medium was incubated overnight in a tri-gas incubator with a 1% O2 concentration. Primary neurons cultured for 5-6 days had their supernatant discarded, were washed twice with pre-warmed PBS, and the original volume of pre-hypoxic DMEM sugar-free medium was added. The cells were then placed in a tri-gas incubator with a 1% O2 concentration for 90 minutes. After hypoxia, the cells were removed, the supernatant discarded, and fresh pre-warmed neuronal culture medium was added for further culture.

[0045] 2.4 NORAD-overexpressing lentivirus infection of mouse primary neurons

[0046] On the second day of primary neuron culture, when changing the medium, take an MOI value of 2 and add NORAD overexpressing lentivirus or control lentivirus to the neuron culture medium according to the following formula:

[0047] Calculate viral load

[0048] Virus dosage per well (μL) = MOI x number of cells at infection / titer (TU / mL) × 1000

[0049] Mix gently. Discard the supernatant, and slowly add the virus-containing culture medium to the cells, avoiding the formation of aerosols. Two days after infection, switch back to normal neuronal culture medium and continue culturing.

[0050] All nozzles and containers that come into contact with the virus must be soaked in 84 disinfectant.

[0051] 2.5 Immunofluorescence staining of neuronal cell smears

[0052] 1) Cell fixation: Discard the cell culture supernatant in the 24-well plate, gently wash the cells twice with 1 mL PBS, add 4% PFA and fix at room temperature for 15 min. Gently wash the cells twice with PBS, add 500 μL PBS to the well plate and use within one week.

[0053] 2) Staining: Aspirate the supernatant PBS from the 4% PFA-fixed slides in the 24-well plate. Add 200 μL of blocking buffer to each well and block at room temperature for 1.5 h. Aspirate the blocking buffer and add 150 μL of primary antibody working solution to each well, incubate at room temperature for 2 h. Discard the supernatant and wash the slides with 500 μL of PBS for 5 min each time, for a total of three times. Aspirate the PBS and add 150 μL of secondary antibody and DAPI staining solution, incubate at room temperature in the dark for 1 h. Discard the secondary antibody and wash the slides with 500 μL of PBS to each well in the dark for 5 min each time, for a total of three times.

[0054] 3) Mounting: Add 30 μL of anti-quenching fluorescent mounting medium to the glass slide, carefully pick up the slide with tweezers, blot off any residual PBS with absorbent paper, fix the slide face down on the glass slide with the mounting medium, being careful not to create air bubbles, air dry at room temperature in the dark for 30 minutes, gently press to remove any residual air bubbles, and store at 4°C in the dark.

[0055] 2.6 Preparation of paraffin-embedded brain tissue specimens

[0056] Mice were anesthetized with isoflurane and fixed in a supine position on a foam board. The thoracic cavity was cut open to expose the heart, and a small notch was cut in the right atrial appendage. A scalp needle was inserted into the left ventricle through the apex of the heart. PBS solution was slowly and evenly injected until the lungs and liver turned white and the rinsing solution no longer showed obvious red. Then, 4% PFA solution was slowly and evenly injected until the mouse's limbs were rigid. The brain tissue was then quickly removed and fixed in 4% PFA solution at 4°C for 48 hours. The brain tissue was rinsed with running water and placed in a paraffin embedding cassette. It was then dehydrated for 15 minutes in four gradients of ethanol (50%, 65%, 75%, and 95%). Then, it was dehydrated in 100% ethanol, with the solution changed three times for 5 minutes each time. The brain tissue was then embedded in paraffin wax at 60°C. The embedded brain tissue was then fixed on a paraffin microtome and sectioned into continuous coronal sections from front to back, with a thickness of 5 μm. The sections contain cortical and hippocampal structures. The sections are placed on a 45°C slide spreader until they are completely flattened. The sections are then placed on a detachable glass slide and finally baked in a 60°C oven for 6 hours before being stored in a slide box.

[0057] 2.7 Nucleocytoplasmic Separation Experiment

[0058] According to PARIS TM Follow the instructions in the kit manual to conduct the experiment. The specific steps are as follows:

[0059] Aspirate the neuronal cell supernatant from a 10cm culture dish, wash once with pre-chilled PBS, and discard the supernatant. Add 300 μL Cell Disruption Buffer, mix on ice, and incubate for 10 min. Scrape cells from the cell culture dish into a 1.5mL centrifuge tube and centrifuge at 500g for 10 min at 4°C. Transfer the supernatant (Section 3) to another 1.5mL centrifuge tube and place on ice; this is the cytoplasmic fraction. Quickly mix the remaining precipitate with 300 μL Cell Disruption Buffer on ice and pipette until clear. Add 300 μL 2×Lysis / Binding Solution, invert the centrifuge tube to mix, add 300 μL ACS-grade anhydrous ethanol, and gently invert the centrifuge tube 3 times. Transfer the sample to a filter column equipped with a collection tube, centrifuge at 10000g for 1 min to pass the sample through the filter column, and discard the filtrate. Add 700 μL Wash Solution 1, centrifuge for 1 min to pass the sample through the filter column, and discard the filtrate. Add 500 μL Wash Solution 2 / 3 to wash the filter column twice, and discard the filtrate. Add 40 μL of Elution Solution preheated to 95℃, centrifuge for 30 s to elute RNA, and collect the eluent into a clean 1.5 mL centrifuge tube. Add 10 μL of Elution Solution preheated to 95℃, centrifuge for 30 s to elute RNA again, and collect the eluent. The collected 40 μL + 10 μL eluent fractions are the nuclear fraction. Measure the RNA concentration in the cytoplasm and nucleus fractions using a UV microspectrophotometer, aliquot, and store at -80℃. Subsequently, reverse transcribe the cytoplasmic and nuclear RNA using a reverse transcription kit, and detect the nuclear proportion of NORAD using quantitative real-time PCR.

[0060] 2.8 Extraction of neuronal cell genomic DNA

[0061] Follow the instructions for the Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304) from Tiangen Biotech (Beijing) Co., Ltd.

[0062] 2.9 RNA extraction from mouse brain tissue and primary neurons

[0063] 1) Source:

[0064] Brain tissue: Right brain tissue of experimental mice was obtained by dissection on ice. The hypothalamus, midbrain and cerebellum were removed. The tissue was placed in a 1.5 mL RNase-free centrifuge tube and 1 mL Trizol was added. The tissue was homogenized until no tissue block was visible to the naked eye.

[0065] Primary neurons: Discard the supernatant of cultured primary neurons, wash twice with PBS, and discard the supernatant. Add 500 μL of Trizol to each well of a 6-well plate, mix well, let stand for 15 min, and collect in a 1.5 mL RNase-free centrifuge tube.

[0066] 2) RNA extraction

[0067] Add 1 mL Trizol / 0.2 mL chloroform to chloroform, vortex for 15 seconds, then invert the centrifuge tube several times to mix. Incubate at room temperature for 3-5 minutes, then centrifuge at 4°C and 12000g for 15 minutes. Transfer the upper aqueous phase to another RNase-free centrifuge tube, add 1 mL Trizol / 0.5 mL isopropanol, vortex for 15 seconds, incubate at room temperature for 10 minutes, then centrifuge at 4°C and 12000g for 10 minutes. Discard the supernatant; white RNA will be visible at the bottom of the tube. Add 1 mL 75% ethanol (prepared with DEPC water) to the tube, vortex gently for 15 seconds. Centrifuge at 4°C and 12000g for 10 minutes, carefully discarding the supernatant. Aspirate excess liquid with a 10 μL RNase-free pipette tip and allow to dry at room temperature for 5-10 minutes. Add 30-50 μL of DEPC water to dissolve the RNA precipitate. Take 1 μL of the solution and measure and record the RNA concentration using an ultra-micro UV spectrophotometer. Aliquot the sample and store it at -80℃.

[0068] 2.10 Real-time quantitative PCR

[0069] 1) Reverse transcription of RNA:

[0070] 2 μL of 5×gDNA Eraser Buffer, 1 μL of gDNA Eraser, 500 ng of RNA sample, and DEPC water were mixed sequentially to form a 10 μL system. The mixture was incubated in a PCR instrument at 42 °C for 2 min to remove genomic DNA. PrimeScript RTEnzyme Mix 1, RT Primer Mix, 5×PrimeScript Buffer 2 (for Real Time), and DEPC water were added sequentially at 1 μL, 1 μL, 4 μL, and 4 μL respectively to the above reaction solution after removing genomic DNA. The mixture was incubated in a PCR instrument at 37 °C for 15 min, followed by denaturation at 85 °C for 5 seconds to obtain cDNA. 180 μL of DEPC water was then added to dilute the cDNA.

[0071] 2) Real-time quantitative PCR:

[0072] Prepare a mixture by adding 0.3 μL of upstream primer (10 μM) and 0.3 μL of downstream primer (10 μM) to each well, along with 5 μL of qPCR SYBR Green Master Mix. After vortexing, incubate on ice. Primer sequences were designed by NCBI and synthesized by Sangon Biotech. See the table below for specific primer sequences used.

[0073]

[0074]

[0075] Add 4.4 μL of cDNA sample and 5.6 μL of the above mixture to each well of a 96-cell real-time PCR plate, seal with a sealing film, and briefly centrifuge for approximately 30 seconds. The reaction program is as follows: Stage 1 pre-denaturation: 95℃ for 5 min, 95℃ for 10 s; Stage 2 cycling reaction: 60℃ for 30 s, 95℃ for 15 s, for 40 cycles; Stage 3 melting curve: 60℃ for 60 s, 95℃ for 15 s. After the reaction, record the Ct value. Calculate the relative expression level of the target gene: using GAPDH (mouse) as an internal reference gene, 2... -△△Ct This is the relative expression level, i.e., ΔCt = target gene Ct value - average internal reference gene Ct value, ΔΔCt = ΔCt - ΔCt 对照组 .

[0076] 2.11 Laser speckle flow imaging for detecting cerebral blood flow

[0077] After anesthetizing the experimental animals with isocyanate by inhalation, the mice were fixed in a prone position. The scalp was disinfected with iodine, and the skin was incised to fully expose the brain. The laser beam from the laser speckle blood flow imaging system was aimed at the mid-brain region of the mouse, and the area supplied by the middle cerebral artery in the left hemisphere was selected as the region of interest. The areas of interest selected in both hemispheres were equal. The exposure time was set to 10 seconds, and blood flow data were continuously collected. The average cerebral blood flow within both regions of interest was used as the analysis object for statistical analysis.

[0078] 2.12 NORAD fluorescence in situ hybridization (FISH) and immunofluorescence double labeling assay

[0079] FISH experiments were performed on primary cultured neurons and paraffin sections of mouse brain tissue.

[0080] The fixation and paraffin sectioning of mouse brain tissue were performed according to step 1) in section 2.6, except that the required fixative and buffer solutions were pretreated with DEPC.

[0081] Remove the PBS from the fixed cells. Add 1 mL of pre-cooled PBS permeabilization buffer containing 0.5% Triton X-100 to each well and incubate at 4°C for 5 min. Discard the permeabilization buffer, then wash the cells with PBS for 5 min each time, repeating this process three times. Boil the sections in antigen retrieval buffer for 15 min and allow them to cool naturally. Then, draw circles with water blocking, add 50 μL of proteinase K (20 μg / ml), and digest at 37°C for 20-30 min. Rinse with pure water, then wash with PBS three times for 5 min each time. Add 200 μL of prehybridization buffer to each well of cells and block at 37°C for 30 min; simultaneously preheat the hybridization buffer at 37°C; under light-protected conditions, add 2.5 μL of 20 μM lncRNA FISH Probe Mix stock solution or internal control FISH Probe Mix stock solution to 100 μL of hybridization buffer; discard the prehybridization buffer in each well of cells, add 100 μL of probe hybridization buffer containing the probe, and hybridize overnight at 37°C in the dark (for paraffin sections, use 50 μL of prehybridization buffer and hybridization buffer). Wash each well of cells three times with 500 μL of hybridization wash buffer I at 42°C in the dark, 5 min each time; wash cells with 500 μL of hybridization wash buffer II at 42°C in the dark for 5 min; wash cells with 500 μL of hybridization wash buffer III at 42°C in the dark for 5 min; wash cells with 500 μL of PBS at room temperature for 5 min (for paraffin sections, use 100 μL of hybridization wash buffer at each step). Cells and paraffin sections were blocked for 1 hour with 200 μL and 50 μL of PBST containing 5% FBS (PBS + 0.3% Triton), respectively. Primary antibodies were diluted in 200 μL and 50 μL of PBS and added to cells and sections, respectively. The sections were incubated overnight at 4°C, followed by washing with PBS 3 × 5 min. Primary antibodies were diluted in 200 μL and 50 μL of PBS and added to cells and sections, respectively. The sections were incubated at room temperature for 1 hour, followed by washing with PBS 3 × 5 min. DAPI staining solution was applied for 10 min in the dark; the sections were then washed three times with PBS, 5 min each time. 30 μL of anti-quenched fluorescent mounting medium was added to each slide, with the cell slides facing down and the paraffin sections covered with a coverslip. Care was taken to avoid air bubbles. The slides were air-dried at room temperature in the dark for 30 min, and any remaining air bubbles were gently pressed out. The slides were then stored at 4°C in the dark. The tissue sections were scanned using an automated tissue scanner, and cell images were acquired using a laser confocal microscope.

[0082] 2.13 Protein extraction from brain tissue and neurons

[0083] After anesthetizing mice, the skull was severed, and the right brain was quickly removed on ice and placed into a 1.5 mL centrifuge tube pre-added with 1 mL of RIPA lysis buffer. Neuronal cells in a 6-well plate were washed twice with pre-chilled PBS, scraped off with a cell scraper, and centrifuged at 500g at 4°C for 5 min. The supernatant was discarded, and 100 μL of RIPA lysis buffer was added to the pellet. The sample was lysed using an ultrasonic homogenizer with the probe inserted into the sample, set to 20% power, and sonicated for 1 second followed by 1 second rest. Brain tissue samples were sonicated approximately 10 times, and neuronal samples approximately once. The samples were placed on ice for 30 min, inverting the centrifuge tube occasionally to mix. The samples were then centrifuged at 14000g for 20-30 min in a pre-chilled 4°C high-speed centrifuge. The supernatant was aliquoted and frozen at -80°C. One sample tube was added to 5×SDS loading buffer, boiled in a metal bath at 100°C for 10 min, and placed on ice for Western blot analysis.

[0084] 2.14 BCA method for determining protein content in brain tissue and neuronal cells

[0085] Diluted standards were added in 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, and 20 μL wells to 96-well plates, respectively. For wells containing less than 20 μL, the corresponding volume of PBS buffer was added to bring the total volume of liquid in each well to 20 μL. 2 μL of the prepared brain tissue sample was added to each 96-well plate, along with 18 μL of PBS buffer, bringing the total volume of liquid in each well to 20 μL. Finally, BCA working solution was added. 180 μL of BCA working solution was added to each well containing standards and sample, and the mixture was gently pipetted to mix. Three blank wells were prepared, each containing only 200 μL of BCA working solution as a blank control. The plates were incubated at 37°C in the dark for 10 min. After the temperature returned to room temperature, the OD value at 562 nm was measured and recorded using a microplate reader. Finally, a standard curve was constructed using Excel, and the protein content in the samples was calculated.

[0086] 2.15 Western blot

[0087] Remove the cooked protein samples and add the markers and samples from each experimental group sequentially. Adjust the volume of the protein markers and blank wells to be equal to the sample volume using 1×SDS loading buffer. Fill the inner tank with 1× protein electrophoresis buffer, and add 1× protein electrophoresis buffer to the outer tank until it covers the platinum wire in the inner tank. First, use a constant voltage of 70V to run the protein samples to the boundary between the separating gel and the stacking gel, then switch to a constant voltage of 120V and continue electrophoresis until the bromophenol blue in the samples reaches the lower edge of the separating gel, then stop electrophoresis. Prepare 1× transfer buffer before starting electrophoresis and store it at 4℃. 30 minutes before the end of electrophoresis, cut a PVDF membrane of the same size as the gel, soak the PVDF membrane in methanol for about 30 seconds, then immerse it in the transfer buffer. At the same time, immerse the sponge and filter paper in the transfer buffer to moisten them. Remove the glass plate, cut off the stacking gel and the portion below bromophenol blue, cut the gel according to the band size of the control protein marker, and place it on filter paper that has been completely soaked in transfer buffer. Create a transfer "sandwich" in the following order: cathode → sponge → filter paper → gel → PVDF membrane → filter paper → sponge → anode. Transfer at a constant current of 150 mA. The specific transfer time depends on the molecular weight of the target protein. Use tweezers to remove the PVDF membrane from the transfer "sandwich" and place it face down in an antibody incubation chamber containing blocking buffer. Incubate at room temperature for 1-2 hours. Discard the blocking buffer and wash the membrane once with TBST. Dilute the antibody to be tested with the primary antibody incubation buffer according to the recommended concentration in the antibody instructions: Bcl2 1:1000, Bax and CaMKIIα 1:2000, Actin 1:5000. Add the primary antibody incubation buffer to the antibody incubation chamber and incubate overnight at 4°C with shaking. Discard the primary antibody incubation solution, add TBST, and wash the PVDF membrane three times (5 min each time) on a shaker with the membrane facing up. Place the PVDF membrane face down, add the hP-labeled secondary antibody diluted 1:5000 with TBST, and gently shake on a shaker at room temperature for about 1 hour. Discard the secondary antibody incubation solution, add TBST, and wash the PVDF membrane five times (10 min each time) on a shaker with the membrane facing up. Mix the chromogenic solutions A and B in a 1:1 ratio, store in the dark, and evenly drop the mixture onto the PVDF membrane. Expose the membrane on an exposure unit and acquire images.

[0088] 2.16 CCK-8 neuron proliferation

[0089] The CCK-8 assay was used to detect the survival of OGD-knockout neurons in NORAD-knockout mice. The specific steps are as follows:

[0090] For neurons seeded into 96-well plates and treated with OGD for 24 hours, add 10 μL of CCK-8 solution to approximately 100 μL of cell culture medium per well and mix gently. Place the culture plate in a CO2 incubator and incubate at 37°C in the dark for 1–4 hours. After incubation, measure the absorbance (OD value) of each well at 450 nm using a microplate reader, record the data, and calculate the cell viability.

[0091] 2.17 Transmission Electron Microscopy Experiment

[0092] Mix 50 mL PBS, 20 mL 10% PFA solution, and 10 mL 25% glutaraldehyde solution thoroughly, then add ddH2O to bring the volume to 100 mL to prepare a 2% PFA-2.5% glutaraldehyde perfusion solution. Perfuse anesthetized mice with this solution, dissect the mice to obtain brain tissue, and cut the required samples into 1×1×1 mm pieces. 3 The slides were placed in electron microscopy fixative and fixed at 4°C for 48 hours. Section preparation, staining, and image acquisition were all performed by Chengdu Lilai Biotechnology Co., Ltd.

[0093] Example 1

[0094] RNA was extracted from the right cerebral cortex and hippocampus of mice 24 hours after HIBD, and NORAD expression was detected by RT-PCR in both the sham-operated and HIBD model groups. Results showed that NORAD expression in brain tissue was significantly decreased 24 hours after HIBD compared to the sham-operated group (P<0.05) (see [link to HIBD model group]). Figure 1 ).

[0095] Example 2

[0096] Brain tissue from 7-day-old SPF-grade C57BL / 6 mice used to construct the HIBD model was analyzed using NORAD fluorescence in situ hybridization (FISH) and immunofluorescence double staining. The results showed that NORAD had almost no co-localization with microglia labeled with ionized calcium-binding adaptor molecule 1 (Iba1), but extensive co-localization with neurons labeled with neuronal nucleoprotein (NeuN) and astrocytes labeled with glial fibrillary acidic protein (GFAP), suggesting that NORAD is primarily localized in neurons and astrocytes (see [link to article]). Figure 2 ).

[0097] Example 3

[0098] Primary neurons cultured for 6 days were divided into two groups: a control group and an experimental group (OGD group). The experimental group underwent oxygen-glucose deprivation (OGD) experiment. Cells were harvested at 1 h, 6 h, 12 h, 24 h, and 48 h after OGD treatment. RNA was extracted, and changes in NORAD at each time point were detected. The results showed that NORAD levels were lower in the control group from 1 h to 48 h after OGD treatment (see...). Figure 3 The differences were statistically significant, with the most significant decrease observed at 24 hours. This indicates that NORAD content in neurons decreased after OGD treatment.

[0099] RNA was extracted from the nucleus of neurons in the control group and the OGD group to detect the distribution of NORAD. The results showed that NORAD was mainly distributed in the cytoplasm before and after OGD treatment. Figure 4 (A) FISH staining of NORAD in neurons revealed that NORAD was mainly distributed in the cytoplasm, and no nuclear-cytoplasmic translocation was observed after OGD treatment. Figure 4 (B in the text). Double immunofluorescence staining with NORAD FISH and Tom20-labeled mitochondria or Lamp1-labeled lysosomes revealed that NORAD co-localized with mitochondria and lysosomes before and after neuronal OGD (OGD). Figure 4 (C in the text) This indicates that NORAD is mainly located in the cytoplasm of neurons, and there is no nucleoplasmic translocation after OGD.

[0100] Example 4

[0101] Brain tissue from P8 NORAD knockout mice (NORAD knockout mice 8 days after birth) was subjected to transmission electron microscopy. Transmission electron microscopy revealed mitochondrial swelling and vacuolation of mitochondrial cristae in NORAD knockout mice neurons (see...). Figure 5 Apoptosis occurred (as indicated by the yellow arrow) (see...) Figure 5 (As indicated by the red arrow) This illustrates the changes in the neuronal microstructure of NORAD knockout mice.

[0102] Further Western blot analysis of the brain tissue from the aforementioned NORAD knockout mice revealed a decrease in phosphorylated receptor-interacting protein kinase 3 (p-Ripk3) and phosphorylated mixed-series protein kinase-like domains (p-MLKL) in P8 NORAD knockout mice. Figure 6 In the A group, cleaved caspase-3 was increased, while Bax / Bcl2 remained unchanged after NORAD knockout. Figure 6 (B in the text) TUNEL staining was performed on paraffin sections of the brains of P18 wild-type mice and P18 NORAD knockout mice (TUNEL kit, Promega). The results showed that NORAD knockout mice exhibited more positive TUNEL fluorescence. Figure 6 C). Co-staining p-MLKL with neuronal nuclear antigen (NeuN) antibody on paraffin sections of the brains of P18 wild-type and P18 NORAD knockout mice revealed reduced p-MLKL positivity in NORAD knockout mice. Figure 6 (D in the text). The above results indicate that NORAD knockout causes damage to neuronal mitochondria, increases apoptosis but reduces necrotic apoptosis.

[0103] Example 5

[0104] RNA was extracted from brain tissues of wild-type and NORAD knockout mice 24 hours after HIBD modeling, and the mRNA levels of Ripk1, Ripk3, and MLKL were detected by RT-PCR. The results showed that the mRNA levels of Ripk3 and MLKL were downregulated in NORAD knockout mice after HIBD modeling, and the differences were highly significant compared with those in wild-type mice (P < 0.01). Figure 7 This indicates that the mRNA levels of Ripk3 and MLKL were reduced in the brain tissue of NORAD knockout mice 24 hours after HIBD.

[0105] Example 6

[0106] Laser speckle imaging was used to detect cerebral blood flow in wild-type and NORAD knockout mice after HIBD modeling. Laser speckle imaging showed that in wild-type mice treated with HIBD, the mean cerebral blood flow in the right hemisphere region of interest was significantly reduced compared to the left hemisphere region of interest. Simultaneously, a comparison of the mean cerebral blood flow in the right hemisphere region of interest between wild-type HIBD mice and NORAD knockout HIBD mice revealed that the mean cerebral blood flow in the right hemisphere region of interest was increased in the NORAD knockout HIBD group compared to the wild-type HIBD group (see...). Figure 8 ).

[0107] Example 7

[0108] Neurons overexpressing control lentivirus and NORAD lentivirus were treated with OGD and DNA was extracted. RT-PCR analysis of mtDNA content in each group revealed that mtDNA content decreased after OGD treatment, while mtDNA content increased after NORAD overexpression (P < 0.01) (see [link to original text]). Figure 9 ).

[0109] Neurons overexpressing control lentivirus and NORAD lentivirus were treated with OGD and then subjected to CCK8 assay. Figure 10 As shown in Figure A, the cell viability of neurons overexpressing NORAD increased after OGD compared to the control group, with statistical results showing P < 0.01. Western blot analysis of protein samples revealed increased Bcl2 protein expression and a decreased Bax / Bcl2 ratio in neurons overexpressing NORAD after OGD, with statistical results showing P < 0.01. Figure 10 (B in the text). RNA samples were collected and analyzed by RT-PCR. Following OGD, the mRNA levels of Ripk1, MLKL, and Ripk3 in neurons overexpressing NORAD increased at different time points. Figure 10(C) Protein samples were collected and analyzed by Western blotting. The results showed that after OGD, neurons overexpressing NORAD showed no significant difference in Ripk1, MLKL, and Ripk3 proteins compared to the control group, but p-MLKL was increased. Statistical results showed P < 0.01 (C). Figure 10 (D in the text). The above results show that OGD-treated neurons exhibit increased activity, decreased apoptosis, and increased necrotic apoptosis after NORAD overexpression.

[0110] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. Application of NORAD in the preparation of drugs for the prevention or treatment of hypoxic-ischemic encephalopathy.

2. The application according to claim 1, characterized in that, The drug is one that promotes NORAD expression.

3. The application according to claim 2, characterized in that, The drug that promotes NORAD expression contains a lentiviral overexpression vector for NORAD.

4. The application according to claim 1, characterized in that, The drug is a drug that promotes the vitality of OGD neurons deprived of oxygen and / or inhibits the death of OGD neurons deprived of oxygen and sugar.

5. The application according to claim 1, characterized in that, The drug is one that upregulates the levels of Ripk3 and MLKL in oxygen-glucose deprived OGD neurons.

6. A drug for the prevention or treatment of hypoxic-ischemic encephalopathy, characterized in that, It contains promoters that enhance NORAD expression.

7. The drug according to claim 6, characterized in that, The drug is a drug that promotes the vitality of OGD neurons deprived of oxygen and / or inhibits the death of OGD neurons deprived of oxygen and sugar.

8. The drug according to claim 6, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The drug according to claim 6, characterized in that, The dosage forms of the drug include capsules, tablets, granules, oral preparations, emulsions, sprays, ointments, or suspensions.

10. A neuronal cell overexpressing NORAD, characterized in that, Lentiviral cells containing NORAD were transfected into cultured neurons in vitro.

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