Use of a circRNA_0004468 in preparation of a drug for treating ischemic stroke
By revealing the circRNA_0004468/miR-1224/VEGFA ceRNA network, the problem of unclear RIPostC neuroprotective mechanism was solved, key regulatory targets were provided, and multi-level validation and clinical translation prospects were realized, promoting the treatment of ischemic stroke.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- THE SECOND AFFILIATED HOSPITAL OF KUNMING MEDICAL UNIV
- Filing Date
- 2026-03-17
- Publication Date
- 2026-06-09
AI Technical Summary
In the existing technology, the neuroprotective molecular mechanism of RIPostC is unclear and lacks key regulatory targets, which limits its precise clinical application and efficacy optimization.
Through transcriptome sequencing and bioinformatics analysis, the ceRNA regulatory network circRNA_0004468/miR-1224/VEGFA was screened and validated, revealing the molecular mechanism of RIPostC neuroprotection and providing a method to achieve neuroprotection by regulating this network.
The system elucidates the gene regulatory network of RIPostC, provides highly specific therapeutic targets, achieves multi-level and multi-model validation, has clear signal transduction and effect pathways, and promotes clinical translation prospects and drug development.
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Figure CN121846124B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the interdisciplinary field of molecular biology and neuromedicine, specifically to a non-coding RNA regulatory network and its application in brain protection, and more specifically, to the mechanism of action of a ceRNA regulatory network composed of circular RNA (circRNA_0004468), microRNA (miR-1224) and vascular endothelial growth factor A (VEGFA) in remote post-ischemic adaptation (RIPostC)-induced brain protection and its application as a therapeutic target. Background Technology
[0002] Ischemic stroke is one of the leading causes of disability and death worldwide. Currently, revascularization therapy (such as intravenous thrombolysis and mechanical thrombectomy) is the gold standard for restoring blood flow, but it is limited by a strict time window (usually 4.5-24 hours) and many contraindications, meaning only a small number of patients benefit from it. Reperfusion itself can also lead to more severe damage, namely ischemia / reperfusion injury. Therefore, exploring safe, effective, and widely applicable neuroprotective strategies, especially methods to activate endogenous protective mechanisms, has become an urgent research need.
[0003] Remote post-ischemic adaptation (RIPostC) is a non-invasive physical intervention method that reduces damage to the primary ischemic organ (such as the brain) by briefly and repeatedly performing ischemia / reperfusion procedures on a distant organ (such as a limb). It has significant advantages such as simplicity, non-invasiveness, cost-effectiveness, and minimal side effects, and holds great promise for clinical application. However, the specific molecular mechanisms by which RIPostC exerts its neuroprotective effect, especially the fine-grained regulatory network at the gene expression level, have not been fully elucidated. This mechanistic uncertainty severely restricts the precise clinical application of RIPostC, the optimization of its efficacy, and the development of related drugs.
[0004] In recent years, non-coding RNAs, especially circular RNAs, have played an increasingly important role in gene regulation. Due to their high stability and strong conservation, circRNAs are considered potential new targets for disease diagnosis and treatment. One of their core mechanisms is that, as competitive endogenous RNAs, they bind to microRNAs through a "sponge-like" binding effect, thereby relieving the inhibition of microRNAs on their target messenger RNAs. In ischemic brain injury, angiogenesis is crucial for rescuing the ischemic penumbra and promoting the recovery of neurological function, and VEGFAs are core factors regulating angiogenesis.
[0005] Although some studies suggest that RIPostC may be related to certain signaling pathways or factors, no research has yet revealed the core molecular switch for RIPostC-induced neuroprotection from the perspective of the systemic ceRNA regulatory network of "circRNA-miRNA-mRNA". Therefore, the key technical problem to be solved in this application is to elucidate the specific ceRNA regulatory network behind the neuroprotective effect of RIPostC, discover the key molecules that play a pivotal role in it, and verify their functions, thereby providing a novel theoretical basis and drug target based on endogenous protective mechanisms for the treatment of ischemic stroke. Summary of the Invention
[0006] This invention aims to address the problems of unclear molecular mechanisms of RIPostC neuroprotection and the lack of key regulatory targets in existing technologies. Specifically, this invention focuses on discovering and validating a ceRNA network that is specifically regulated in the RIPostC process and is crucial for neuroprotective effects, thereby laying the foundation for developing novel neuroprotective agents or adjunctive therapies targeting this network.
[0007] To address the aforementioned technical problems, this invention, through transcriptome sequencing and bioinformatics analysis, combined with an in vitro oxygen-glucose deprivation / recombinant oxygen model and an in vivo rat cerebral ischemia / reperfusion model, has for the first time screened and validated a novel ceRNA regulatory network: the circRNA_0004468 / miR-1224 / VEGFA axis. This invention provides an application of this novel ceRNA regulatory network in remote post-ischemic adaptation (RIPostC) brain protection and in an in vitro ischemia / reperfusion injury model. This network consists of circRNA_0004468, miR-1224, and VEGFA. Specifically, circRNA_0004468 acts as a "molecular sponge" for miR-1224, competitively binding to miR-1224, thereby relieving the post-transcriptional inhibition of miR-1224 on its target gene VEGFA, ultimately upregulating VEGFA expression. Based on the above findings, this invention also provides a method and application for achieving brain protection by regulating the elements in the ceRNA network. For example, by upregulating circRNA_0004468 or downregulating miR-1224, it is possible to promote VEGFA expression, activate downstream protective signals, and thus reduce nerve damage.
[0008] The beneficial effects of this invention are:
[0009] Compared with the prior art, the present invention has the following significant advantages:
[0010] (1) A novel endogenous protective mechanism was elucidated for the first time: This invention, for the first time, systematically revealed the molecular mechanism by which RIPostC exerts its neuroprotective effect from the perspective of ceRNA regulatory networks, and discovered a novel regulatory axis of circRNA_0004468 / miR-1224 / VEGFA. This discovery provides a clear and specific gene regulatory map for understanding the complex physiological process of RIPostC, and advances the mechanism research from macroscopic pathways to the precise RNA molecular interaction level.
[0011] (2) Provides highly specific therapeutic targets: This invention clarifies that circRNA_0004468 and miR-1224 are two key modifiable nodes in the regulatory network.
[0012] circRNA_0004468: As a protective molecule, its overexpression in cell models (OGD / R) can significantly improve cell viability, reduce apoptosis, and upregulate PI3K / VEGFA expression; in animal models, it can synergistically enhance the neuroprotective effect of RIPostC (such as further reducing neurological function scores and reducing infarct volume).
[0013] miR-1224: As a damage-related molecule, its inhibition produces a protective effect similar to that of circRNA_0004468 overexpression in both in vivo and in vitro models. These two molecules have great potential as novel drug targets, and agonists (e.g., mimics, overexpression vectors) or inhibitors (e.g., antagonists, siRNAs) can be designed, respectively.
[0014] (3) It achieves full verification across multiple levels and models: The conclusions of this invention are not based on a single experiment, but are supported by a complete and rigorous chain of evidence:
[0015] Targeting relationship verification: Dual-luciferase reporter gene assays directly confirmed the targeted binding of circRNA_0004468 to miR-1224, and miR-1224 to VEGFA 3'UTR.
[0016] Spatial colocalization: FISH experiments showed that the two colocalized in the cytoplasm, providing a physical basis for the "sponge adsorption" phenomenon.
[0017] In vitro functional validation: In the OGD / R cell model, gain-of-function / loss-of-function experiments confirmed that manipulating this network can directly regulate cell survival, apoptosis, and the expression of downstream protective genes.
[0018] In vivo functional validation: In the tMCAO rat model, intervention with this network significantly enhanced the positive effects of RIPostC on neurological function, infarct volume, and angiogenesis markers (VEGFA, CD31, CD34).
[0019] This invention elucidates a clear signal transduction and effector pathway: it goes beyond simply discovering interactions and preliminarily elucidates downstream pathways. Experimental data show that this ceRNA network upregulates VEGFA, thereby activating the classic pro-survival signaling pathway PI3K / AKT, and ultimately promoting angiogenesis (increased CD31+ and CD34+ cells), explaining the protective effect from the molecular signaling to tissue repair level.
[0020] (4) The clinical translation prospects are clear and diverse:
[0021] Diagnostic biomarkers: The levels of circRNA_0004468 or miR-1224 in the blood may serve as non-invasive biomarkers for assessing stroke risk, RIPostC efficacy, or prognosis.
[0022] Treatment strategies: Develop gene therapies (such as overexpressing lentiviruses) targeting circRNA_0004468 or nucleic acid drugs (such as antagonists) targeting miR-1224, which can be used as independent neuroprotective therapies or in combination with RIPostC to enhance efficacy.
[0023] Drug screening platform: Using this network as a target, a high-throughput screening model can be established to find small molecule compounds that can specifically activate this pathway. Attached Figure Description
[0024] Figure 1 Dual-luciferase reporter gene assay to verify the targeting relationship between circRNA_0004468 and miR-1224;
[0025] Figure 2 Dual-luciferase reporter gene assays validated the targeting relationship between miR-1224 and VEGFA 3'UTR;
[0026] Figure 3 FISH experiments showed that circRNA_0004468 and miR-1224 were co-localized in the cytoplasm of PC12 cells;
[0027] Figure 4 : CCK-8 assay to detect the effects of regulatory circRNA_0004468 or miR-1224 on the viability of OGD / R model PC12 cells;
[0028] Figure 5 Flow cytometry was used to detect the effects of regulating circRNA_0004468 or miR-1224 on apoptosis in OGD / R model PC12 cells;
[0029] Figure 6qRT-PCR was used to detect the expression level of miR-1224 in PC12 cells of each group;
[0030] Figure 7 qRT-PCR was used to detect the expression level of circRNA_0004468 in PC12 cells of each group;
[0031] Figure 8 qRT-PCR was used to detect the mRNA expression level of PI3K in PC12 cells of each group;
[0032] Figure 9 qRT-PCR was used to detect the mRNA expression level of VEGFA in PC12 cells of each group;
[0033] Figure 10 Western blot was used to detect the protein expression levels of PI3K, p-PI3K and VEGF in PC12 cells of each group.
[0034] Figure 11 Effects of different intervention groups on the Zea-Longa score of neurological deficits in rats with cerebral ischemia / reperfusion.
[0035] Figure 12 Effects of different intervention groups on Ludmila Belayev scores of neurological deficits in rats with cerebral ischemia / reperfusion.
[0036] Figure 13 Effects of different intervention groups on the percentage of cerebral infarction volume in rats with cerebral ischemia / reperfusion.
[0037] Figure 14 Immunohistochemical detection of VEGF protein expression and distribution in rat brain tissue of different intervention groups;
[0038] Figure 15 Immunofluorescence assay was used to detect the expression of CD31 protein in the brain tissue of rats in different intervention groups.
[0039] Figure 16 Immunofluorescence assay was used to detect the expression of CD34 protein in the brain tissue of rats in different intervention groups.
[0040] Figure 17 qRT-PCR was used to detect the expression level of circRNA_0004468 in the brain tissue of rats in different intervention groups;
[0041] Figure 18 qRT-PCR was used to detect the expression level of miR-1224 in the brain tissue of rats in different intervention groups;
[0042] Figure 19qRT-PCR was used to detect the mRNA expression level of VEGFA in the brain tissue of rats in different intervention groups;
[0043] Figure 20 Western blot was used to detect the protein expression level of VEGF in the brain tissue of rats in different intervention groups. Detailed Implementation
[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be described in more detail below with reference to the accompanying drawings.
[0045] Plasmid construction and lentivirus packaging
[0046] In this invention, the rat circRNA_0004468 sequence (SEQ ID NO: 1) and VEGFA sequence (SEQ ID NO: 3) were synthesized by Nanjing Genscript Biotech Co., Ltd., and cloned into the GPLVX-CircRNA_Mini-Puro and PGMLV-CMV-MCS-3×Flag-PGK-Puro lentiviral vectors, respectively, by treatment with the same restriction endonuclease as the lentiviral vector. The constructed lentiviral vector and its helper packaging vector plasmid were co-transfected into HEK-293T cells, and the cell supernatant rich in lentiviral particles was collected and concentrated to obtain a high-titer lentiviral concentrate.
[0047] Dual-luciferase reporter gene assay
[0048] Based on the binding regions of circRNA_0004468 and VEGFA to miRNA-1224, wild-type plasmids (circRNA_0004468-WT), mutant plasmids (circRNA_0004468-MUT), wild-type VEGFA plasmids (VEGFA-WT), and mutant plasmids (VEGFA-MUT) were constructed. Using liposome transfection, circRNA_0004468-WT, circRNA_0004468-MUT, VEGFA-WT, and VEGFA-MUT luciferase reporter gene plasmids were co-transfected into 293T cells with miRNA-1224 mimic and a mimic control (NC mimic). After 48 hours of routine culture, the activities of fireflies and renin-luciferase in each well were measured using an ELISA reader, following the instructions of the dual-luciferase reporter gene assay kit.
[0049] The results are as follows Figure 1As shown, in cells transfected with circRNA_0004468-WT, the relative luciferase activity of the miRNA-1224 mimic group was lower than that of the NC mimic group (P < 0.0001); in cells transfected with circRNA_0004468-MUT, there was no significant difference in relative luciferase activity between the miRNA-1224 mimic group and the NC mimic group. These results indicate that circRNA_0004468 can target and regulate miRNA-1224.
[0050] The results are as follows Figure 2 As shown, in VEGFA-WT transfected cells, the relative luciferase activity of the miRNA-1224 mimic group was lower than that of the NC mimic group (P < 0.05); in VEGFA-MUT transfected cells, there was no significant difference in relative luciferase activity between the miRNA-1224 mimic group and the NC mimic group. These results indicate that VEGFA is a target gene of miRNA-1224.
[0051] Fluorescence in situ hybridization (FISH) assay to detect the localization of circRNA_0004468 and miRNA-1224 in cells.
[0052] Log-phase PC12 cells were seeded onto 24-well slides. When the cell confluence reached 60%, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. After washing three times with PBS, proteinase K was added and the cells were digested and permeated at 37°C for 5 min. After washing three more times with PBS, the in situ hybridization detection kit (Guangzhou Exon Biotechnology) was used. The novel_circRNA_0004468 probe (with CY3 tag) and rno-miRNA-1224 probe (with FAM tag) used in the experiment were synthesized by Shanghai Sangon Biotech.
[0053] The results are as follows Figure 3 As shown, circRNA-0004468 and miR-1224 are co-localized in the cytoplasm of PC12 cells.
[0054] Lentiviral transfection, miRNA mimics / inhibitors transfection, siRNA transfection experiments
[0055] Lentiviral transfection methods
[0056] Inoculate 1 mL (3 × 10) 4PC12 cells were suspended in 12-well plates and cultured for 24 hours. The medium was then changed to DMEM high-glucose basal medium containing a mixture of Polybrene infection reagent and lentivirus. After transfection for 24 hours, the medium was changed to DMEM high-glucose complete medium and cultured for another 24 hours before proceeding with subsequent experimental procedures.
[0057] miRNA mimics / inhibitors, siRNA transfection methods
[0058] Inoculate 1 mL (3 × 10) 4 PC12 cells (1 cell per well) were suspended in 12-well plates. When the cell confluence reached approximately 50%, the medium was changed to contain the transfection complex (according to RiboFECT). TM According to the instructions of the CP transfection kit, prepare DMEM high-glucose basal medium (miRNA mimics / inhibitors or siRNA transfection complex), and perform subsequent experimental procedures 48 h after transfection.
[0059] Methods for constructing a glucose-oxygen deprivation / replenishment glucose-oxygenation cell model
[0060] Inoculate 1 mL (3 × 10) 4 (Number of cells) The target cell suspension was placed in a 12-well plate and cultured overnight. After the cell culture medium was changed to 1 mL of glucose-free basal medium, the cells were subjected to hypoxia treatment in a three-gas incubator for 2 h. The hypoxia conditions were 3% O2, 5% CO2, and 92% N2. After the hypoxia was completed, the cell culture medium was changed to DMEM high-glucose basal medium and reoxygenated for 24 h. The reoxygenation conditions were 20.9% O2 and 5% CO2 in a normal two-gas incubator.
[0061] CCK8 Experiment
[0062] PC12 cells were seeded at 2500 cells / well in 96-well plates and cultured overnight. The medium was then changed to DMEM high-glucose basal medium containing lentivirus, miRNA mimics / inhibitors, and siRNA for transfection. After transfection, the cells were subjected to glucose-oxygen deprivation / replenishment treatment. After cell treatment, 10 μL of CCK8 reagent was added to each well and the cells were incubated at 37°C for 2 h. The absorbance (OD value) of the cells at 450 nm was measured using a microplate reader.
[0063] The results are as follows Figure 4As shown, compared with the normal group, the cell viability of the glucose-oxygen deprivation group and the glucose-oxygen deprivation / re-glucose-oxygen group was reduced; compared with the glucose-oxygen deprivation / re-glucose-oxygen group, upregulation of miR-1224 (miR-1224 mimics) or downregulation of circRNA_0004468 (circRNA_0004468 si-RNA) further reduced cell viability; downregulation of miR-1224 (miR-1224 inhibitors) or upregulation of circRNA_0004468 (circRNA_0004468 overexpression) increased cell viability compared with the glucose-oxygen deprivation / re-glucose-oxygen group.
[0064] Apoptosis experiment
[0065] Collect target cells, wash with PBS, resuspend cells in 100 μL of flow cytometry apoptosis binding buffer, add 5 μL Annexin V-FITC staining solution and 5 μL PI staining solution, mix well, incubate at room temperature in the dark for 15 min, then add PBS to bring the liquid level to 1 mL, and determine the number of apoptotic cells by flow cytometry.
[0066] The results are as follows Figure 5 As shown, compared with the normal group, the proportion of apoptotic cells was increased in the glucose-oxygen deprivation group and the glucose-oxygen deprivation / re-glucose-oxygen group; compared with the glucose-oxygen deprivation / re-glucose-oxygen group, upregulation of miR-1224 (miR-1224 mimics) or downregulation of circRNA_0004468 (circRNA_0004468 si-RNA) could further increase the proportion of apoptotic cells; downregulation of miR-1224 (miR-1224 inhibitors) or upregulation of circRNA_0004468 (circRNA_0004468 overexpression) resulted in a lower proportion of apoptotic cells than in the glucose-oxygen deprivation / re-glucose-oxygen group.
[0067] Real-time quantitative PCR detection of RNA expression levels
[0068] Target samples were collected, and total RNA was extracted from cells / tissues using the Trizol method. cDNA was synthesized using a reverse transcription kit. The target gene was amplified using a qRT-PCR kit, with 2... -△△Ct The method calculates the relative expression of the target gene.
[0069] The primer sequences used in the experiment are as follows:
[0070] Rat β-actin_F: TATGGAATCCTGTGGCATC; Rat β-actin_R: GTGTTGGCATAGAGGTCTT.
[0071] Rat PI3K_F: GGTGATTGAGAAGTGTAA; Rat PI3K_R: CATTGGATAGGACTGTAG.
[0072] Rat VEGFA_F: CATGAAGTGGTGAAGTTC; Rat VEGFA_R: GTACTCCTGGAAGATGTC.
[0073] Primers for miR-1224 and circRNA_0004468 were purchased from RiboBio.
[0074] The results are as follows Figure 6 As shown, in cell experiments, compared with the normal group, the expression level of miR-1224 was increased in the glucose-oxygen deprivation group and the glucose-oxygen deprivation / recombined glucose-oxygen group; compared with the glucose-oxygen deprivation / recombined glucose-oxygen group, upregulation of miR-1224 (miR-1224 mimics) or downregulation of circRNA_0004468 (circRNA_0004468 si-RNA) could further increase the expression level of miR-1224; downregulation of miR-1224 (miR-1224 inhibitors) or upregulation of circRNA_0004468 (circRNA_0004468 overexpression) resulted in a decrease in the expression level of miR-1224 compared with the glucose-oxygen deprivation / recombined glucose-oxygen group.
[0075] The results are as follows Figure 7 As shown, in cell experiments, compared with the normal group, the expression level of circRNA_0004468 was decreased in the glucose-oxygen deprivation group and the glucose-oxygen deprivation / re-glucose-oxygen group; compared with the glucose-oxygen deprivation / re-glucose-oxygen group, upregulation of circRNA_0004468 (circRNA_0004468 overexpression) led to an increase in circRNA_0004468 expression, while downregulation of circRNA_0004468 (circRNA_0004468 si-RNA) led to a decrease in circRNA_0004468 expression; upregulation of miR-1224 (miR-1224 mimics) or downregulation of miR-1224 (miR-1224 inhibitors) had no significant effect on the expression of circRNA_0004468.
[0076] The results are as follows Figure 8As shown, in cell experiments, compared with the normal group, the gene expression level of PI3K was reduced in the glucose-oxygen deprivation group and the glucose-oxygen deprivation / re-glucose-oxygen group; compared with the glucose-oxygen deprivation / re-glucose-oxygen group, upregulation of miR-1224 (miR-1224 mimics) or downregulation of circRNA_0004468 (circRNA_0004468 si-RNA) further reduced the gene expression level of PI3K; downregulation of miR-1224 (miR-1224 inhibitors) or upregulation of circRNA_0004468 (circRNA_0004468 overexpression) increased the gene expression level of PI3K compared with the glucose-oxygen deprivation / re-glucose-oxygen group.
[0077] The results are as follows Figure 9 As shown, in cell experiments, compared with the normal group, the gene expression level of VEGFA was reduced in the glucose-oxygen deprivation group and the glucose-oxygen deprivation / re-glucose-oxygen group; compared with the glucose-oxygen deprivation / re-glucose-oxygen group, upregulation of miR-1224 (miR-1224 mimics) or downregulation of circRNA_0004468 (circRNA_0004468 si-RNA) further reduced the gene expression level of VEGFA; downregulation of miR-1224 (miR-1224 inhibitors) or upregulation of circRNA_0004468 (circRNA_0004468 overexpression) increased the gene expression level of VEGFA compared with the glucose-oxygen deprivation / re-glucose-oxygen group.
[0078] The results are as follows Figure 17 As shown, in animal experiments, compared with the sham-operated group, the expression level of circRNA_0004468 in the brain tissue of rats in the model group was increased, suggesting that it may be involved in the pathophysiological response after cerebral ischemia-reperfusion. Compared with the model group, RIPostC intervention further upregulated the expression of circRNA_0004468, and this upregulation was further enhanced when combined with overexpression of circRNA_0004468, inhibition of miR-1224, or overexpression of VEGFA, indicating that these treatments can synergistically promote the expression of circRNA_0004468. It is worth noting that the increase in circRNA_0004468 expression caused by the combination of RIPostC and the above interventions is consistent with the observed trend of neuroprotective effect, suggesting that the upregulation of circRNA_0004468 may be an important link in the endogenous protective mechanism, and its further increase in expression level may be related to the enhanced neuroprotective effect.
[0079] The results are as follows Figure 18As shown, in animal experiments, compared with the sham-operated group, the expression level of miR-1224 in the brain tissue of rats in the model group was reduced, suggesting that it may be involved in the pathological process after cerebral ischemia-reperfusion. Compared with the model group, RIPostC intervention could more significantly downregulate the expression of miR-1224; and when RIPostC was combined with overexpression of circRNA_0004468, direct inhibition of miR-1224, or overexpression of VEGFA, the inhibitory effect on miR-1224 expression was further enhanced, as evidenced by a further reduction in miR-1224 expression. This more significant inhibition of miR-1224 expression is consistent with the observed enhanced neuroprotective effect, suggesting that RIPostC and its combined intervention may more effectively inhibit miR-1224, a potential damaging factor, thereby more fully relieving its inhibitory effect on downstream protective pathways (such as VEGFA signaling), ultimately achieving a better neuroprotective effect.
[0080] The results are as follows Figure 19 As shown, in animal experiments, the expression level of the VEGFA gene in the brain tissue of rats in the model group was higher than that in the sham-operated group, which may be the endogenous vascular repair and compensatory response initiated after cerebral ischemia injury. Compared with the model group, RIPostC intervention can further upregulate VEGFA expression, suggesting that RIPostC can actively enhance this protective response. When combined with overexpression of circRNA_0004468, inhibition of miR-1224, or direct overexpression of VEGFA on the basis of RIPostC, the expression of VEGFA gene was increased more significantly, and its level was higher than that of the RIPostC intervention group alone. As a key angiogenesis and neuroprotective factor, the expression level of VEGFA is closely related to tissue repair, angiogenesis, and cell survival. Therefore, the more significant increase in VEGFA induced by the above combined intervention means that the activation of angiogenesis and neuroprotective pathways is more fully enhanced, which explains at the molecular level why the combined strategy can produce a superior neuroprotective effect.
[0081] Western blotting is used to detect protein expression levels.
[0082] Collect the target sample, add RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, lyse on ice, and determine the protein concentration using the BCA method with the supernatant; add loading buffer and incubate at 100℃ for 10 min to denature the protein; then perform SDS-PAGE gel electrophoresis, transfer membrane, block, add primary antibodies (PI3K antibody 1:1000, p-PI3K antibody 1:1000, VEGF antibody 1:1000), incubate overnight at 4℃, wash the membrane, incubate with secondary antibody at room temperature for 2 h, develop, and analyze the protein bands using ImageJ software.
[0083] The results are as follows Figure 10 As shown, compared with the normal group, the protein expression levels of p-PI3K and VEGF were decreased in the glucose-oxygen deprivation group and the glucose-oxygen deprivation / re-glucose-oxygenation group. Compared with the glucose-oxygen deprivation / re-glucose-oxygenation group, upregulation of miR-1224 (miR-1224 mimics) or downregulation of circRNA_0004468 (circRNA_0004468 si-RNA) further decreased the protein expression level of p-PI3K, and upregulation of miR-1224 (miR-1224 mimics) further decreased the protein expression level of VEGF; downregulation of miR-1224 (miR-1224 inhibitors) or upregulation of circRNA_0004468 (circRNA_0004468 overexpression) resulted in higher protein expression levels of p-PI3K and VEGF than in the glucose-oxygen deprivation / re-glucose-oxygenation group.
[0084] The results are as follows Figure 20 As shown, compared with the sham-operated group, the expression level of VEGF protein in the brain tissue of rats in the model group was increased, which may be a compensatory response of endogenous vascular repair and neuroprotection triggered after ischemic injury. Compared with the model group, RIPostC intervention further upregulated the expression of VEGF protein, indicating that this intervention can actively enhance such protective responses. When RIPostC was combined with overexpression of circRNA_0004468, inhibition of miR-1224, or direct overexpression of VEGFA, the increase in VEGF protein expression was more significant, and its level was higher than that of the RIPostC intervention group alone. Since VEGF is a core factor in promoting angiogenesis, improving blood perfusion, and supporting neuronal survival, the stepwise increase in its expression level—especially under combined intervention conditions—means that the vascular repair and neuroprotective pathways were activated more strongly and persistently. Therefore, the "further increase" in VEGF expression brought about by these treatments functionally corresponds to a more significant angiogenesis-promoting and neuroprotective effect, thus providing a key molecular explanation for the synergistic effect of the combined strategy.
[0085] Lateral ventricle localized injection of lentivirus and miRNA antago
[0086] After anesthetizing the rats, their heads were prepared, and they were fixed in a prone position on a stereotaxic apparatus. Following routine disinfection, a midline incision was made in the head, and the fascia and periosteum were bluntly dissected to expose the skull. The rat's brain was leveled anteriorly and posteriorly. The injection site was located at the anterior fontanelle, 1.5 mm lateral to and 1 mm posterior to the anterior fontanelle, marking the coordinates for lateral ventricle injection. At the marked points, a 1 mm diameter drill bit was used to open the skull, and a microsyringe was inserted 3.5 mm downwards to inject lentivirus or miRNA antago.
[0087] Rat model of cerebral ischemia / reperfusion and experimental methods for distal ischemic adaptation (RIPostC)
[0088] Methods for establishing a rat model of cerebral ischemia / reperfusion: Rats were fasted for 12 hours before surgery but allowed free access to water. After anesthesia, rats were fixed in a supine position on a temperature-controlled surgical board. Routine skin preparation and disinfection were performed. A midline incision was made in the neck, and subcutaneous tissue was bluntly dissected to expose the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The distal and proximal ends of the ECA were ligated, with a slipknot tied at the proximal end. Arterial clamps were used to clamp the proximal ends of the CCA and ICA. The middle segment of the ECA was cut, and a suture plug was inserted. The suture plug branched through the CCA and entered the ICA, then along the ICA into the middle cerebral artery (MCA), with an insertion depth of approximately 18-20 mm. The suture plug was fixed, and the skin was sutured. After 2 hours of ischemia, the suture plug was slowly removed to reperfusion, restoring blood flow to the MCA. The ECA incision was then ligated. After the rats regained consciousness, they were allowed free access to food and water.
[0089] Methods for adaptation experiment after distal ischemia: Before reperfusion, the bilateral femoral arteries of rats were exposed. During reperfusion, the bilateral femoral arteries were clamped with arterial clamps. The bilateral femoral arteries were blocked for 10 minutes and released for 10 minutes. This was repeated three times.
[0090] Behavioral evaluation methods
[0091] Behavioral evaluations were performed on rats 48 hours after reperfusion.
[0092] Neurological function was assessed using the Zea-Longa scoring system: 0 points, no neurological deficit symptoms; 1 point, the contralateral forepaw cannot be fully extended when the tail is lifted; 2 points, the body rotates to the contralateral side; 3 points, the body tilts to the contralateral side; 4 points, the animal cannot walk spontaneously and is accompanied by a decline in consciousness; 5 points, the animal dies.
[0093] The Ludmila Belayev scoring system was used to assess the motor function and sensory integrity of rats. This included two parts: the postural reflex test and the limb placement test. The postural reflex test, also known as the tail-lift test, scored as follows: 0 points for no obvious neurological deficits, 1 point for flexion of the contralateral limb due to infarction, and 2 points for a positive lateral push test. The limb placement test included visual, tactile, and proprioceptive subtests. The first two were tested from the front and side, respectively, while the latter was tested only from the front. A normal limb placement response was scored as 0 points, a delayed response but not exceeding 2 seconds as 1 point, and a delayed response exceeding 2 seconds as 2 points. The total score ranged from 0 to 12 points, with higher scores indicating more severe functional impairment.
[0094] The results are as follows Figure 11As shown, compared with sham surgery, the Zea-Longa score of rats in the model group was significantly increased. Compared with the model group, RIPostC intervention, RIPostC intervention with simultaneous overexpression of circRNA_0004468, RIPostC intervention with simultaneous inhibition of miR-1224, or RIPostC intervention with simultaneous overexpression of VEGFA significantly reduced the Zea-Longa score of rats, among which the RIPostC intervention with simultaneous overexpression of circRNA_0004468 group had the lowest Zea-Longa score.
[0095] The results are as follows Figure 12 As shown, compared with sham surgery, the Ludmila Belayev score of rats in the model group was significantly increased; compared with the model group, RIPostC intervention, RIPostC intervention with simultaneous overexpression of circRNA_0004468, RIPostC intervention with simultaneous inhibition of miR-1224, or RIPostC intervention with simultaneous overexpression of VEGFA significantly reduced the Ludmila Belayev score of rats.
[0096] Methods for measuring cerebral infarction volume
[0097] After the behavioral evaluation of rats was completed, the rats were anesthetized and perfused with pre-cooled physiological saline via cardiac perfusion. Brain tissue was then harvested and immediately frozen at -80℃ for 20 minutes. After removal, the brain tissue was transversely and evenly divided into 5 sections along the coronal plane, from the olfactory bulb to the cerebellum. The sections were immersed in 2% TTC staining solution and incubated at 37℃ in the dark for 15 minutes. Ischemic brain tissue appeared white, while normal brain tissue was stained red. The stained sections were then removed and fixed in 4% paraformaldehyde at room temperature for 30 minutes before photography. ImageJ software was used to calculate the infarct area and total area of the brain sections, according to the formula: Percentage of infarct volume = (Sum of white ischemic areas in each section) / (Sum of total areas of each brain section) × 100%.
[0098] The results are as follows Figure 13 As shown, compared with sham surgery, the percentage of cerebral infarction volume in the model group rats was significantly increased. Compared with the model group, RIPostC intervention, RIPostC intervention with simultaneous overexpression of circRNA_0004468, RIPostC intervention with simultaneous inhibition of miR-1224, or RIPostC intervention with simultaneous overexpression of VEGFA could significantly reduce the percentage of cerebral infarction volume in rats. Moreover, overexpression of circRNA_0004468, inhibition of miR-1224, or overexpression of circRNA_0004468 could further enhance the neuroprotective effect of RIPostC.
[0099] Immunohistochemistry and immunofluorescence
[0100] Paraffin section preparation: After anesthetizing rats, the brain tissue was quickly removed by perfusion of pre-cooled physiological saline into the heart and immediately fixed in 4% paraformaldehyde for 72 hours. Paraffin sections were prepared by dehydration, clearing, paraffin impregnation, embedding, sectioning, mounting, and drying.
[0101] Immunohistochemical staining was performed, followed by dewaxing of paraffin sections to water, antigen retrieval, blocking with hydrogen peroxide in the dark, blocking with sheep serum, incubation with primary antibody (VEGF antibody 1:100) at 4°C overnight, rinsing with PBST, incubation with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 30 min, rinsing with PBST, followed by DAB staining solution and incubation at room temperature in the dark for 5-15 min, termination of staining with distilled water, counterstaining with hematoxylin, differentiation with 1% hydrochloric acid alcohol, dehydration with graded alcohols, clearing with xylene, mounting with neutral resin, and observation under a regular optical microscope.
[0102] Immunofluorescence staining was performed, paraffin sections were dewaxed to water, antigen retrieval was performed, sheep serum was used for blocking, primary antibodies (CD31 antibody 1:200, CD34 antibody 1:150) were incubated overnight at 4°C, washed with PBST, fluorescent secondary antibody was incubated at room temperature in the dark for 1 hour, washed with PBST, mounted with anti-fluorescence quencher containing DAPI, and observed under a fluorescence microscope.
[0103] The results are as follows Figure 14 As shown, compared with sham surgery, the expression level of VEGF protein in the brain tissue of rats in the model group was significantly increased. Compared with the model group, RIPostC intervention, RIPostC intervention with simultaneous overexpression of circRNA_0004468, RIPostC intervention with simultaneous inhibition of miR-1224, or RIPostC intervention with simultaneous overexpression of VEGFA could further increase the expression level of VEGF protein in rat brain tissue. Moreover, the expression level of VEGF protein was higher in the RIPostC intervention group than in the RIPostC intervention group alone.
[0104] The results are as follows Figure 15 As shown, compared with sham surgery, the expression level of CD31 protein in the brain tissue of rats in the model group was significantly increased. Compared with the model group, RIPostC intervention, RIPostC intervention with simultaneous overexpression of circRNA_0004468, RIPostC intervention with simultaneous inhibition of miR-1224, or RIPostC intervention with simultaneous overexpression of VEGFA could further increase the expression level of CD31 protein in rat brain tissue. Moreover, the expression level of CD31 protein was higher in the RIPostC intervention group than in the RIPostC intervention group alone.
[0105] The results are as follows Figure 16As shown, compared with sham surgery, the expression level of CD34 protein in the brain tissue of rats in the model group was significantly increased. Compared with the model group, RIPostC intervention, RIPostC intervention with simultaneous overexpression of circRNA_0004468, RIPostC intervention with simultaneous inhibition of miR-1224, or RIPostC intervention with simultaneous overexpression of VEGFA could further increase the expression level of CD34 protein in rat brain tissue. Moreover, the expression level of CD34 protein was higher in the RIPostC intervention group than in the RIPostC intervention group alone.
[0106] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements without departing from the principles of the present invention, and these improvements should also be considered within the scope of protection of the present invention.
Claims
1. The application of circRNA_0004468 in the preparation of a drug for treating ischemic stroke, characterized in that, The sequence of the circRNA_0004468 consists of SEQ ID NO:
1.
2. The application according to claim 1, characterized in that, The drug works by upregulating the expression of circRNA_0004468.