CN117224560A - Use of LncRNA - Google Patents
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- CN117224560A CN117224560A CN202311268014.7A CN202311268014A CN117224560A CN 117224560 A CN117224560 A CN 117224560A CN 202311268014 A CN202311268014 A CN 202311268014A CN 117224560 A CN117224560 A CN 117224560A
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Abstract
The invention relates to the technical field of biology, in particular to application of LncRNA, and specifically relates to application of LncRNA with a sequence shown as SEQ ID NO:1 in the preparation of medicaments for treating cerebral apoplexy. The invention can reduce the pre-LncRNA, which is beneficial to avoiding the occurrence of cerebral ischemia/reperfusion injury, has a protective effect on the nerve injury of focal cerebral ischemia reperfusion mice in the acute phase, can be popularized and applied in the preparation of the medicines for treating ischemic cerebral apoplexy, has good application prospect, provides a direction and a technical basis for searching and researching and developing new medicines for exactly and effectively treating cerebral vascular diseases such as ischemic cerebral apoplexy, and is beneficial to early break away from affliction of cerebral vascular diseases patients such as ischemic cerebral apoplexy.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of LncRNA.
Background
With the deep research of genetic engineering, scientists have shown great interest in developing drugs by using genetic engineering. Long non-coding RNAs (LncRNA) are a class of RNA molecules that exist in the transcriptome of some eukaryotic cells, greater than 200 nucleotides in length, and are not translatable to proteins. Most LncRNA lack an effective open reading frame, however, they can undergo splicing, capping, and polyadenylation. LncRNA is a byproduct of RNA polymerase ii transcription because it cannot be translated into protein, and is initially considered a byproduct of gene transcription, which is considered to be unwanted "noise". However, after confirming that LncRNA Xist is involved in the regulation of X chromosome inactivation in 1991, lncRNA is gradually attracting attention of researchers and has received a great deal of attention. Many studies have been carried out in succession to indicate that LncRNA is directly or indirectly involved in regulating various biological processes, such as apoptosis, differentiation, angiogenesis, proliferation, etc., and that it plays an important role in various diseases such as breast cancer, lung cancer, cerebral stroke, heart failure, etc. The secondary and tertiary structures of LncRNA are relatively conserved compared to their primary structures, while LncRNA perform its broad biological functions by virtue of post-transcriptional modification to form secondary structures such as stem loops and hairpin. LncRNA plays its functional role by interacting with other molecules (DNA, RNA, proteins, etc.). Scientists have demonstrated that the nucleotide sequence of LncRNA is highly conserved across species, with very low mutation rates, expressed with gene clustering phenomena, space-time and tissue specificity. The LncRNA regulates the expression profile of protein at the posttranscriptional level to determine the progress and diversity of a series of important vital activities such as cell differentiation, embryo development and the like, provides a theoretical basis for revealing the disease occurrence mechanism from the aspect of RNA regulation, and provides a new gene target and molecular marker for disease diagnosis and treatment.
On the other hand, cerebral stroke (cerebral stroke) is an acute cerebrovascular disease, and a specific pathogenesis is derived from a disease in which cerebral blood vessels are suddenly ruptured or blood cannot flow into the brain due to blood vessel blockage, and cerebral tissue injury is caused, which is commonly called as "stroke". World health organization has reported that stroke is the second leading factor in worldwide mortality, causing at least 600 tens of thousands of deaths each year. In developed countries, stroke is also a major cause of permanent disability, leading to economic loss in the health industry. By 2030, the mortality rate of stroke is predicted to be over 12% worldwide. In China, cerebral apoplexy is also a main cause of permanent disability, and economic loss of the health industry is caused. Cerebral apoplexy mainly comprises ischemic cerebral apoplexy and hemorrhagic cerebral apoplexy, and the ischemic cerebral apoplexy accounts for about 75% of the morbidity. Currently, regardless of whether intravenous drug thrombolysis or arterial mechanical thrombolysis is used, even if cerebral blood supply is restored within a short period of time, the occurrence of cerebral ischemia/reperfusion injury is unavoidable. Therefore, there is an urgent need to find and develop new therapeutic agents that are exactly effective.
Disclosure of Invention
The invention aims to solve the technical problem of providing LncRNA and application thereof in preparing medicaments for treating cerebral apoplexy.
In order to solve the problems, the invention provides LncRNA, the sequence of which is shown as SEQ ID NO:1, and provides the application of the LncRNA in preparing medicaments for treating cerebral apoplexy, which can be beneficial to the treatment of cerebral apoplexy.
The invention also provides application of the recombinant plasmid containing LncRNA in preparing a medicament for treating cerebral apoplexy, which is characterized in that the LncRNA has a sequence shown in SEQ ID NO: 1.
As an improvement of the technical scheme, the medicine is an oral medicine or an injection medicine. Further, the medicament is administered to a subject after symptoms of ischemic stroke.
As an improvement of the technical scheme, the medicine is used for ischemic cerebral apoplexy.
As an improvement to the above-described solution, the LncRNA mediates activation of NF- κb signaling pathway by physically binding to iκbα and promoting its phosphorylation, so that neuroinflammation and neuronal apoptosis occur during cerebral ischemia reperfusion.
As improvement of the technical scheme, the medicine can reduce the expression level of mRNA of TNF-alpha, IL-6, IL-1 beta Bax, caspase-3 and Caspase-9 and improve the expression level of mRNA of Bcl-2.
As an improvement of the technical scheme, the construction method of the recombinant plasmid comprises the following steps:
the implementation of the invention has the following beneficial effects:
the invention discovers a novel ischemia reperfusion induced LncRNA (SEQ ID NO: 1) by analyzing a rat focal cerebral ischemia reperfusion model; the LncRNA showed elevated levels of expression in middle cerebral artery occlusion/reperfusion (MCAO/R) mouse brain tissue and oxygen-glucose deprivation/reoxygenation (OGD/R) treated rat cerebral cortex primary neurons. The results suggest that LncRNA mediates activation of NF- κb signaling pathway by physically binding to iκbα and promoting its phosphorylation, leading to neuroinflammation and neuronal apoptosis upon cerebral ischemia reperfusion; in addition, lncRNA is down-regulated to serve as an NF- κB inhibitor in the pathological processes of OGD/R and MCAO/R, so that the LncRNA is suggested to have a protective effect on cerebral ischemia reperfusion injury, is applied to the preparation of medicines for treating ischemic cerebral apoplexy, is favorable for avoiding the occurrence of cerebral ischemia/reperfusion injury, and has good clinical application market prospect. LncRNA plays an important role in neuronal inflammation and apoptosis by activating NF- κB signaling pathway, and is a promising therapeutic target for ischemic cerebral apoplexy. Further experimental related studies show that LncRNA can reduce the expression of inflammatory factors such as TNFa, IL6 and the like in the brain, thereby exerting a therapeutic effect on ischemic stroke, and the optimal use time of LncRNA for treatment is applied after the symptom of the ischemic stroke of a treated subject. LncRNA can be applied to the preparation of oral medicines and injection medicines for treating ischemic cerebral apoplexy and other measures for treating ischemic cerebral apoplexy.
Drawings
FIG. 1 is a graph of neurological scores using mNSS after MCAO/R surgery;
FIG. 2 is a graph showing the results of quantitative analysis of TTC-stained brain tissue sections and cerebral infarction volumes of mice;
FIG. 3 is a representative image of anti-neun immunohistochemical detection of ischemic penumbra nerve cell loss;
FIG. 4 is a graph of TUNEL staining analysis of ischemic penumbra apoptosis;
FIG. 5 is a graph showing the results of detection of mRNA levels of inflammatory apoptosis factors;
FIG. 6 is a graph showing the results of detection of the level of inflammatory apoptosis factor protein.
Detailed Description
The present invention will be described in further detail below in order to make the objects, technical solutions and advantages of the present invention more apparent.
The invention provides an application of lncRNA in preparing a medicine for treating ischemic cerebral apoplexy, wherein the LncRNA has the following sequence: 5' -acacggcaca agatggccgg agaggcagca gcaagcccag ctgtcaggcg gtctgccgcg gcccgtgggc tcgccgctgc gctgggagcg ctaccccggc gtcctggagg gctctgggagagcggggagg ccgcggctgg ggcgaggcgg ccgcgcgcga tgtgggagct cgcggccgga gcgcccggggaggccgggcc cacgacgccc gtggctccgc tgcggcagcg gcggtgctgg tgtcgcgcgc ggccgggaggcggcttcgcg ccgcgggcgg gaggctgcgg cgggcgaccc gtcctggaca cgcgaggaag agcgagccgatggcggcagg ggccgcgctt cgacccggta acttagaaga tgataattaa tgtggttgct gataattctgaataaataca gcttttatcc caggtgtgcc attttgaaga ctgagaccat agagttctaa gaataaaggaaagagccctt gggaaattat tatatatagc aaaaatgtga atcctcagat ggaatgaaag gcctgcacca
tagacatcga agcatttaca ccccgcttga agagtttgaa atggacttta ccactgagaa atcaagatgg
cagcccatta tggggaattg aggaaaatgg attaatgcaa gaatgctgta atattataca accaacacag
gattctttta atgtggattc catgaaatga atgattctta cccaacacaa atggacagtg gaatttactt cctaaagact
tgttacatgt catgtacatt tttgacatct ggagaagact ctacaattct acaaatggta gtttgtattc ctggaatttc
ttgcagtttg atctgaagtg accttatgga atgttaactt taataaaatc tctaaaactt aaaaatttct cagtttgggc
cttttataaa ttttttcagt ggacgttttc attaaatgtt cattttacct tgataacatt ttgaccttta tagctgctgc
ttttcattaa agtatactaa ataaagctgg taaaatggaa tggttttgtt tgtttgggat ttatgtgtat tagaaaacct
ggatttttcc cgtgattaag tggaatggtc ctttggttaa aatagatata ttctctctcc cccttccccc tttcttctat
ttaaaaaaaa aattattttt atttattttt tattttttca ggacagggtt tctctgtgta tccttggctg tcctgcaact
ctgtctgtag accagccagg cctggaattt gcagtgatct tcctgcctct gcctcctgag tgctgggatt aaagctatat
acacccagtt tggctggctt tttattatag ggtttgaata aattcctttg agtttgggtc atttcaaaat atcttgcatt
ttaatcattg ggtaatggta actttgaatg tgtcaggaga ttatagtgaa tatataatat aaaacaaagt gctggtctca
tttcctgtat agtccaagac agtctcagtg tctttcaatt tttgttgatg aaagtcgtcc tttgccatgt gaaatgctca
tctgaatatt tgagttatac taatttgtat tgtattgtct tatattagag gtgtattttt aaagtctttg ggtatacatt
aaagggctta cctaatatga cagtgtaatt tatttacatt tgaaaaaaat tcttgttttt cttgttttgt gcagttggga
gtttaaatga attataccat gctttagatt ggttacccat ggttgaaatc aaatgccaga ttttcattga ttgctacttt
ggtaataaat ttgataaagc agtgatgggg actttgttga ccaaatgacg attggtggtg tttttctcag tatatagaac
aaagaatgaa atagtctgtt attaatgtaa actaccttag aatacacatc tgtggttaat acagatgcag tgtttgttct
ttggatgatg tgaggtggtt gatatttgta aattgaaaac ttgagttttt ggattttgat ggaaatatgt ttgtagactt
gaaactgatt ttttacattt tcttagattt aacataatta cgtttggtag tgaaactaaa atgaattcgt ttgctggagt
gtcatggttt tgaccagtag ggttcagtaa aggaatgttt ttagtttttt cggcatttgt atgtggttgg ttacttgtgt
gcatgggaga gaatactttg ttgcatttgt aattttgttt ctatagacct cgtgcttcca tctggagggg gtttgtcttt
gacagttgat aattggctgg tagatgtggc ctatttaagt ggcggtgatt gatgaaatgt gagtgtactt agaattcgat
ttcaaacatg agtgaaaggg ttcttttgat tttggagttg aggttgagtg tttgatttaa aggtgaccaa ggttggtgta
atttcttgaa ttcgaagtta ttaattatgc ccaaatatat gtaagaagta aatatgtttg atgtgtggca cttccaagat
taatggttta caaagaagtt tgtttcttct gggggggaaa atggttgtat taacttttgc tctactgcag ttgaacacta
gggaggcgct cttgagttgt aaaaaattac cccttccttc cagttgaggt ggaaaattcc cccttaatct ttagatgttt
ttaggcacca taaagaaggc aagagttgat ttaatttatg gtgttgaaat gttgcttcta tttacaagcc attttaccca
tggtggggtg ttaattattt taaagattag ttcttaaact gagctttaga gtctcttgaa attatcttta tttcaaaatg
gcttgataaa actccacttc ctgtccaggg aggaagttat tttagctcaa ggactgaaat atttagcaga tctttagaca
aaaggaacaa attacatttc tctctggctt aaaaagggtg cttgagccag agggggttaa gaagcccttc
cccctccctc tgatggagtg gtctggaggg gcagggaggt gtggagttgg gggtcttctg gctctcttgg
atggagaatc tggttctttt tctttctttc tttctttttt tttttttttt ggtggagatg aaggggtggg tctatggtac
atcacctgag ttgtggggta aatgtagaga gtgtcaatca aaggcagagc tctcagagct gggaaggagg
ctctagatgg cggctgtgcc ttagagagag cgcgctttgc tccctgcttc ggttcacttt acgcaacttt ccctaacttt
cgggcagcct cagggggccc ccgcagcccc ctgcctcttc tagtgactta ctggggtcga tccgaacctt
tttttgggag aaaagctgct tttaggagct ttcttttcgt gccttgttgg aagaagctgc cgtattgaga gcccaggtcg
ttgttttttc cagcttagaa gccatggcgc acctccattt ttgtgcgctc tcctaatgag gttttttttt ccccttccgg
acctgtttta gtattaatta ttgctttatt ttttgacaag tcaacatatt tgaggattat ttcatttact ttttgttttt
taacggagaa ttttgcgttt ttaaaaatta ttttggatcg gatatttttc tactactaga taggactctt gctttggacc
tactacatgg atcaggtaag tccatgataa tcagtgataa ttttaaaaaa attaatgttg aacttggctt tatttactgt
aaatatgaga aattcctgga tgacgcaata agatagagag attgttattt gtatccagtg tgttactttt gagaaacttt
ccttattgat agtgatgctt ctgtcgttca gtttatcccc ctctcatttt aaaacggaaa aatagaaata ttttgttttt
ctggtgttta tcgttgttgc attacatgaa tatcgctaac atcgtcagga ctttgttcct gataataaaa ccatccgcag
aagctgggac ctcacctttc tttcaacttt tgacagtaaa tacctgggca caggacttca aagcaaacac
agattccccc tcccccttaa tatttaagaa ttaaaagatg atgagaaata aggacaaaag ccaagaggag
gacagttcgc tacacagcaa tgcatcgagg tatgagctat caactttctt tctttctttc tttttttttt tttttcggga
aacctcagct tacacttgtt tactttgaca gacatggatt gctgtctgta tttcaagaat tttctaactt gggttggttt
ttaaagcgtg accttgagat tgctaacatg ccacaccctg attttttggt atcaatttat gaaagttgag ttgtgactta
aggcctctta cagcattcag aaggtaatgt acttttctcc ggaggggcct ttcctagaaa ctgtagtttt tctgtcagta
aattttcagg ttttaggttt tcatgctgca gaaaaatgac cttgtctgtt tagccattgc tgcattgcac cattttacat
acaaggcaaa tttttttctt tgaagaatct gggatattca aacttgtaac cgagggccat agagcagcct
ggtagtagta aattatagta aatagacatt tgagaaagtt atatgaatga aaaaatgtac atttaaaaac acaatcacaa
ataggacccc ttgactgatg cctttcagtt tatatttttg attgtttctt ttgatacacg tgacaattaa aatttttttt
atgacttatc aaaaccttaa taaatagttc ctgagatcat taaatctgta accacaaaaa ttttggaatg ctattaatac
ttaagctttg tgtttgttac tatggcaatg taattgtatt ttttatgctt aaaatattac aaggtttatg tttgtgtata
ttaagacagt tctttctggt tgataataat tgtattttat tttatcctta tgttttagtt ggtgctgctt taaatgtatc
catttatttt gacagtgtcc tgtaatgctt tctcttctga agctttttaa tgtgctgttt gtttaaatca tgatttctcc
ttcattcttt gaagtagctt atcagtggct gaagtaataa agcatataag ataaaaaaaa aaaaaaa-3’(SEQ ID
NO: 1). The LncRNA or the recombinant plasmid thereof is applied to the preparation of oral medicines or injection medicines for treating cerebral arterial thrombosis.
The present invention was also verified by the following test.
1. Preparation of focal cerebral ischemia reperfusion animal model
The modified Longa-wire-plug method is used for preparing a model of cerebral middle arterial embolism (middle cerebral artery occlusion, MCAO) of a mouse. C57BL/6 male mice, 7-8 weeks old, weighing 18-21g, were kept in a clean-grade environment. Firstly, the skin of the neck of a mouse is cleaned by a shaver to expose complete skin tissues, then the skin is carefully cut by surgical scissors, a surgical microscope is started, the tissue around the neck of the mouse in the visual field is carefully spread by thicker forceps under the microscope to avoid mechanical damage, at the moment, the tracheal muscles are separated to expose the Common Carotid Artery (CCA), the muscles and fascia adhered to the common carotid artery are further peeled off by the micro forceps, the bifurcation of the CCA is visible upwards, and the Internal Carotid Artery (ICA) and the External Carotid Artery (ECA) are continuously visible forwards. At this time, the ligation is performed by the ECA near the bifurcation of the proximal CCA, and the suture plug can be manually circumvented and erroneously inserted into the pteropalatine artery (PPA), so that the ligation is not performed on PPA. Then cutting a small opening on the blood vessel by micro-shearing at the position close to the ligation of the CCA, inserting a nylon wire plug with the head end coated with silica gel into the CCA by 0.18mm through the small opening, ligating the middle end of the blood vessel by a slipknot before the wire plug is inserted to avoid massive hemorrhage of the artery when the wire plug is inserted, loosening the slipknot, slightly pushing to enable the wire plug to advance along the trend of the internal carotid artery, continuing to insert along the trend of the ICA blood vessel until the Middle Cerebral Artery (MCA) of the mouse after passing through the intersection of the ECA and the ICA, and stopping after the wire plug is perceived to be slightly resistant, wherein the distance of the wire plug entering the MCA is about 12-14 mm calculated from the bifurcation, so that the blood flow of the brain is provided by the middle cerebral artery of the mouse is basically blocked. After 60min of ischemia, slowly drawing out the thrombus, ligating the external carotid artery, loosening the silk thread at the common carotid artery to restore the blood flow of the right carotid artery, suturing the incision, sterilizing with iodophor, placing in a cage, feeding at room temperature of 25 ℃, and ending the MCAO to complete the focal cerebral ischemia reperfusion model. The whole process adopts a laser Doppler blood flow instrument to monitor local cerebral blood flow so as to ensure that the model preparation is successful. Before the start of the surgery and before the animals wake up, the body temperature of the mice was maintained at 36.5 ℃ ±1 ℃ using a thermometer. After anesthesia of the sham animals, only the internal and external carotid artery bifurcation was exposed, without the insertion of a plug.
2. Experimental grouping and administration
C57BL/6 male mice were weighed and randomly divided into 4 groups: sham surgery group (Sham), cerebral ischemia model group (Vehicle), intervention negative control group (siRNA-NC), intervention treatment group of LncRNA (siRNA-LncRNA).
The administration method comprises the following steps: according to the weight of the mice, 10% chloral hydrate (4.0 mL/kg) was injected intraperitoneally for anesthesia, and the prone position was fixed, so that the skull was kept level in the middle. Shearing, sterilizing with iodophor, exposing cranium top, dipping proper amount of H 2 O 2 The top of the cranium is rubbed to expose the bregma. The left ventricle coordinate is found according to the brain map of the mouse as follows: anterior-posterior direction = 0.8mm, medial-lateral direction = ±1.4mm, dorsi-ventral direction = 3.5mm relative to bregma. Plasmids containing siRNA-LncRNA or siRNA-NC were slowly injected into each lateral ventricle (3. Mu.L per hemisphere, injection rate 0.15. Mu.L/min, injection time exceeding 15 minutes) and needle was left for 5min after injection was complete. After slowly lifting back to take out the microsyringe needle, the bone wax seals the drill hole. The mice were removed from the stereotactic apparatus and the wound was sutured, and the iodophor sterilized and returned to the feeder cage.
The construction method of the plasmid containing siRNA-LncRNA comprises the following steps: the hairpin structure was amplified with the following primer pair, and the purified amplification product was subjected to overnight ligation with psiLentGeneTM vector at 4℃under the action of T4 DNA ligase, transformed into DH 5. Alpha. And subsequently developed resistance selection using mainly Amp and plate-based culture. DNA was extracted from positive clones, digested with EcoRV, and identified by agarose gel electrophoresis. Positive recombinant plasmids were successfully screened.
Forward primer 5'-AAGACAGTCTCAGTGTCTTTCAATT-3' (SEQ ID NO: 2)
Antisense strand 5'-AATTGAAAGACACTGAGACTGTCTT-3' (SEQ ID NO: 3).
Wherein, the plasmid containing siRNA-NC refers to psiLentGeneTM plasmid.
3. Neuromotor function scoring
The neurological function of mice with ischemia 1h reperfusion 24h was scored accordingly using Longa pentad. The scoring criteria were: 0 point: the mice have no neurological function deficiency and no disturbance of activity; 1, the method comprises the following steps: the contralateral forelimb and forepaw of the mouse cannot be fully extended; 2, the method comprises the following steps: the mice walk involuntarily to the contralateral surgery; 3, the method comprises the following steps: the mice are involuntarily dumped to the opposite side of the operation when walking; 4, the following steps: the mice were severely impaired in walking, had a reduced level of consciousness or were in a comatose state. Wherein, neuro-behavioural score > 1 is divided into success models. In animal experiments, excessive bleeding occurs during the operation; after operation, respiratory abnormality, premature death and sacrifice are all abandoned, and the rejected animals are randomly supplemented in later experiments. The experimental results are shown in fig. 1, and the neuromotor score of the LncRNA-administered intervention treatment group is significantly lower than that of the model group, indicating that LncRNA can significantly improve the neuromotor dysfunction of mice after cerebral ischemia reperfusion.
4. Determination of cerebral infarct volume
The mice were subjected to head breaking for 24h after ischemia reperfusion, brain tissues were rapidly removed, olfactory bulb, cerebellum and low brain stem were removed, and frozen at-20℃for 20min. The brain tissue of the mice was then serially sectioned from the frontal pole at a distance of 3mm from the frontal pole, from front to back, and cut into 5 slices at equal distance. The brain sections were turned over from time to time in 1% TTC incubated at 37℃for 10min to ensure uniform staining. After TTC staining, normal tissues appear red and infarcted tissues appear white. The mouse brain slices were scanned with Image J software and the percentage of infarct volume to the contralateral brain volume was calculated. As shown in fig. 2, the LncRNA treated group had significantly reduced cerebral infarction volume compared to the model group and the negative control group.
5. Immunohistochemical staining
Drying slices in 65 deg.C oven, dewaxing with xylene and alcohol gradient, washing with PBS buffer solution at least 3 times every 5min, and dripping 3%H 2 O 2 The reaction was kept away from light for 20min to inactivate endogenous peroxidase and washed at least 3 times every 5min with PBS buffer. Immersing the slices in 0.01M citrate buffer solution (pH 6.0) heated to boiling by microwaves, repairing for 15min at constant temperature by microwaves, and naturally cooling to room temperature. After blocking the nonspecific antigen with 10% goat serum, primary antibody was added dropwise and incubated overnight at 4 ℃. The next day the primary antibody was washed 3 times 5min each with 0.01M PBS, followed by dropwise addition of secondary antibody and oven incubation at 37deg.C for 1h, and the secondary antibody was washed 3 times 5min each with 0.01M PBS. And dropwise adding Diaminobiphenyl (DAB) for developing in dark, dehydrating, sealing, and observing under a mirror to obtain the film. As shown in FIG. 3, after 24 hours of cerebral ischemia reperfusion, the number of neurons and cells in the cerebral cortex of the mice in the model group and the negative control group were significantly reduced as compared with the sham operation groupNuclear shrinkage; after LncRNA intervention treatment, the quantity of neurons in the cortex of the mice is obviously increased compared with that of a model group, and the morphology of the neurons is also restored.
6. TUNEL staining
Neuronal apoptosis was detected by TUNEL (terminal deoxynucleotidyl transferase mediated dUTP end marker) assay. Cell or brain tissue samples were fixed on glass slides with ice-cold 4% glutaraldehyde for 10 minutes followed by permeabilization with 0.1% (v/v) Triton X-100 (Sigma) for 5 minutes. Slides were incubated with TUNEL incubation mixture for 1.5 hours at 37 ℃ under a wet dark environment. The nuclei were then stained with 4, 6-diphenylamine-2' -phenylhydrazine hydrochloride (DAPI). TUNEL positive staining was observed and photographed using a fluorescence microscope. Apoptosis index refers to the percentage of TUNEL positive cells (positive cells/100% total cells). As shown in fig. 4, after 24 hours of cerebral ischemia reperfusion, neuronal apoptosis was significantly increased in the cerebral cortex of mice in the model group and the negative control group compared with the sham operation group; MCAO/R-induced neuronal apoptosis can be significantly reduced following LncRNA intervention.
7. Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using TRIzol reagent (general Biotech) according to the manufacturer's protocol. The transcription reaction was carried out at 16℃for 30 minutes, followed by incubation at 42℃for 30 minutes and enzyme inactivation at 85℃for 5 minutes. Rapid quantitative PCR was performed using SYBRH Select Master Mix (Vazyme). These transcription reactions were performed using the following parameters: incubate for 30min at 16 ℃, 30min at 42 ℃, 5min at 84 ℃. qRT-PCR reactions were performed using the following parameters: incubation was performed for 2 minutes at 95℃followed by 40 cycles, wherein incubation was performed for 10 seconds at 95℃and 20 seconds at 60 ℃. All results were normalized to GAPDH expression. By using 2 -ΔΔCt Quantitative analysis is carried out by a technical method. All primer sequences used are as follows:
as shown in FIG. 5, 24 hours after cerebral ischemia reperfusion, the mRNA expression levels of pro-inflammatory factors (TNF-alpha, IL-6, IL-1 beta) and pro-apoptotic factors (Bax, caspase-3, caspase-9) in the mouse cerebral cortex tissues of the model group and the negative control group were significantly increased and the mRNA expression levels of anti-apoptotic factors (Bcl-2) were significantly decreased compared with the sham group; after LncRNA intervention treatment, the mRNA expression level of pro-inflammatory factors (TNF-alpha, IL-6 and IL-1 beta) and pro-apoptotic factors (Bax, caspase-3 and Caspase-9) are obviously reduced and the mRNA expression level of anti-apoptotic factors (Bcl-2) is obviously increased.
8. Western blotting (Western blotting)
Mice were anesthetized by intraperitoneal injection with 10% chloral hydrate solution, sacrificed at breaks, and brains were quickly removed on ice, separating the hemispheres of the brain into the injured and non-injured sides. Taking fresh brain tissue in the peripheral area of the injury side infarction, fully homogenizing the brain tissue and the lysate according to the mass volume ratio of 1:10, placing the homogenate into an ice box, fully lysing for 30min on a shaking table, centrifuging for 15min at 4 ℃, absorbing supernatant, adding 6 x protein loading buffer solution according to the volume ratio, denaturing for 15min at 100 ℃, sub-packaging and preserving at-80 ℃. Taking out and dissolving the protein sample at-80 ℃, centrifuging by a centrifuge, adding the protein sample into a lane by a microsyringe, performing SDS-polyacrylamide constant pressure gel electrophoresis separation, and transferring to a PVDF membrane by an electrotransport system. 10% skim milk powder-TBST is sealed for 1-2 h at room temperature. Add primary antibody to 4 ℃ and shake overnight. The next day the primary antibody was discarded, the membrane was washed 10min X4 times with TBST, horseradish peroxidase-labeled secondary antibody was added, incubated at room temperature for 1h on a shaker, rinsed with TBST and developed with ECL luminescent substrate. The Tanon 5200 fully automated chemiluminescence imaging analysis system develops and analyzes. And performing semi-quantitative analysis on the ratio of the target protein gray level value to the internal reference GAPDH gray level value. As shown in fig. 6, 24 hours after cerebral ischemia reperfusion, the mRNA expression levels of pro-inflammatory factors (TNF- α, IL-6, IL-1β) and pro-apoptotic factors (Bax, caspase-3, caspase-9) were significantly increased and the protein expression levels of anti-apoptotic factors (Bcl-2) were significantly decreased in the mouse cerebral cortex tissues of the model group and the negative control group compared to the sham group; after LncRNA intervention treatment, the protein expression level of pro-inflammatory factors (TNF-alpha, IL-6 and IL-1 beta) and pro-apoptotic factors (Bax, caspase-3 and Caspase-9) are obviously reduced and the protein expression level of anti-apoptotic factors (Bcl-2) is obviously increased.
From the above, the invention can reduce the pre-LncRNA, which is beneficial to avoiding the occurrence of cerebral ischemia/reperfusion injury, has a protective effect on the nerve injury of a focal cerebral ischemia reperfusion mouse in the acute phase, can be popularized and applied in preparing the medicine for treating ischemic cerebral apoplexy, has good application prospect, provides a direction and a technical basis for searching and researching and developing new medicine which is exactly effective in treating cerebral vascular diseases such as ischemic cerebral apoplexy, and is beneficial to early break away from pain of cerebral vascular diseases patients such as ischemic cerebral apoplexy.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, such changes and modifications are also intended to be within the scope of the invention.
Claims (6)
- Application of LncRNA in preparing medicament for treating cerebral apoplexy is characterized in that the LncRNA has a sequence shown in SEQ ID NO: 1.
- 2. The application of the recombinant plasmid containing LncRNA in preparing a medicament for treating cerebral apoplexy is characterized in that the LncRNA has a sequence shown in SEQ ID NO: 1.
- 3. The use according to claim 1 or 2, wherein the medicament is an oral medicament or an injectable medicament.
- 4. The use according to claim 1 or 2, wherein the medicament is a medicament for ischemic stroke.
- 5. The use of claim 1 or 2, wherein the LncRNA mediates activation of NF- κb signaling pathway by physically binding to and promoting phosphorylation of ikbα to cause neuroinflammation and neuronal apoptosis upon cerebral ischemia reperfusion.
- 6. The use of claim 1 or 2, wherein the medicament reduces the expression level of mRNA of TNF- α, IL-6, IL-1 βbax, caspase-3, caspase-9, and increases the expression level of mRNA of Bcl-2.
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