Application of m1A methylase TRMT10C in preparation of medicine for treating cerebral arterial thrombosis

Abstract

The invention belongs to the technical field of gene drugs, and particularly relates to application of m1A methylase TRMT10C in preparation of drugs for treating cerebral arterial thrombosis, it is found that expression of the m1A methylase TRMT10C in a cerebral arterial thrombosis model is remarkably increased, and silence of the m1A methylase TRMT10C can effectively improve neurological function scores, reduce cerebral infarction volume and inhibit neuronal apoptosis; and oxidative stress related indexes (for example, NOX2 is reduced, and HO-1 / Nrf2 is increased) are remarkably regulated and controlled. The m1A methylase TRMT10C plays a role in neuroprotection through an Nrf2 / HO-1 signal channel, and has good targeting property and treatment potential. In addition, the TRMT10C inhibitor can be prepared into oral or injection medicines, and is suitable for acute-stage intervention of clinical cerebral arterial thrombosis patients. The invention provides a new target spot and a research and development direction for precise treatment of cerebrovascular diseases, and has important clinical application value and social significance.

Description

Technical Field

[0001] This invention belongs to the field of gene drug technology, specifically relating to the application of m1A methyltransferase TRMT10C in the preparation of drugs for treating ischemic stroke. Background Technology

[0002] Stroke is one of the leading causes of disability and death worldwide, especially ischemic stroke, which accounts for more than 75% of all stroke cases. Although existing treatments such as intravenous thrombolysis and mechanical thrombectomy can restore blood flow to some extent, they cannot effectively curb secondary neurological damage caused by reperfusion injury, resulting in limited clinical efficacy and unsatisfactory patient prognosis.

[0003] In recent years, epitranscriptomics studies have shown that RNA methylation modification plays a key role in neurological diseases. m1A methylation modification, as an important form, has been shown to be involved in the pathological process of cerebral ischemia-reperfusion injury.

[0004] TRMT10C, as an m1A methyltransferase, has had its role in mitochondrial tRNA modification and oxidative stress response preliminarily revealed, but its specific functional mechanism in cerebral ischemia remains unclear, and there has been no systematic research or development of it as a therapeutic target.

[0005] Therefore, it is urgent to conduct in-depth research on the mechanism of action of m1A methyltransferase TRMT10C in ischemic stroke, verify its feasibility and effectiveness as a therapeutic target, provide theoretical basis and technical support for the development of novel neuroprotective drugs, and fill the gap in current clinical treatment strategies. Summary of the Invention

[0006] To address the aforementioned issues, this paper presents the application of m1A methyltransferase TRMT10C in the preparation of drugs for treating ischemic stroke. In vitro and in vivo experiments have demonstrated that silencing m1A methyltransferase TRMT10C significantly improves neurological function and reduces brain injury. Its mechanism is closely related to the activation of the Nrf2 / HO-1 antioxidant pathway, suggesting that m1A methyltransferase TRMT10C may serve as an effective target for treating ischemic stroke.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides the application of m1A methyltransferase TRMT10C as a therapeutic target for stroke.

[0009] The present invention also provides the use of an inhibitor that inhibits the functional expression of m1A methyltransferase TRMT10C in the preparation of a drug for treating stroke.

[0010] As described above, the nucleic acid sequence of the m1A methyltransferase TRMT10C is shown in SEQ ID NO.1.

[0011] Furthermore, in the application described above, the inhibitor is an shRNA that targets and silences the expression of m1A methyltransferase TRMT10C; the nucleic acid sequence of the shRNA is shown in SEQ ID NO.2.

[0012] The present invention also provides a pharmaceutical composition comprising an effective amount of an m1A methyltransferase TRMT10C inhibitor and a pharmaceutically acceptable carrier for the treatment of ischemic stroke.

[0013] Furthermore, the carrier is one or more of chitosan, cholesterol, nanoparticles, and liposomes.

[0014] Furthermore, the pharmaceutical composition as described above is in the form of an oral formulation or an injectable formulation.

[0015] The present invention also provides the application of a reagent for in vitro detection of the expression level of m1A methyltransferase TRMT10C in the preparation of a diagnostic kit for ischemic stroke.

[0016] Furthermore, in the applications described above, the reagent includes specific primers or probes targeting the m1A methyltransferase TRMT10C.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects:

[0018] 1. This invention reveals for the first time the crucial role of m1A methyltransferase TRMT10C in the development and progression of ischemic stroke. Analysis of a focal cerebral ischemia-reperfusion model in SD rats revealed that m1A methyltransferase TRMT10C is a novel ischemia-reperfusion-induced mRNA, with significantly increased expression in brain tissue of rats with middle cerebral artery occlusion / reperfusion (MCAO / R) and neurons treated with oxygen-glucose deprivation / reoxygenation (OGD / R). Experiments confirmed that silencing m1A methyltransferase TRMT10C can significantly reduce oxidative stress damage, improve neurological deficits, reduce infarct volume and neuronal apoptosis by activating the Nrf2 / HO-1 signaling pathway, thereby exerting a therapeutic effect on ischemic stroke. Downregulation of m1A methyltransferase TRMT10C has a clear protective effect against cerebral ischemia-reperfusion injury and can serve as a novel therapeutic target for ischemic stroke. This invention provides experimental evidence and clinical application prospects for the development of oral and injectable drugs targeting m1A methyltransferase TRMT10C, and has significant scientific value and clinical translational potential.

[0019] 2. Based on the above mechanism, this invention further provides the application of inhibitors (such as siRNA or shRNA) targeting the m1A methyltransferase TRMT10C in the preparation of drugs for treating ischemic stroke. These inhibitors have high specificity and good biocompatibility, and can be efficiently delivered through various routes of administration (such as intravenous injection, local administration, etc.), showing promising clinical application prospects. The implementation of this invention will provide new ideas for the precision treatment of ischemic stroke, possessing significant scientific research value and potential market application potential. Attached Figure Description

[0020] Figure 1 This invention provides a neurological scoring chart using mNSS after MCAO / R surgery.

[0021] Figure 2 This is a diagram showing TTC-stained brain tissue sections and quantitative analysis of cerebral infarction volume in SD rats, based on the present invention.

[0022] Figure 3 These are representative images of the pathological findings in ischemic penumbra cells detected by HE staining, NeuN immunohistochemical staining, and TUNEL staining in this invention.

[0023] Figure 4 This is a quantitative statistical diagram showing the detection of ischemic penumbra cytopathology using NeuN immunohistochemical staining and TUNEL staining, as presented in this invention.

[0024] Figure 5 This is a graph showing the mRNA levels of genes related to downstream signaling pathways mediated by the Nrf2 / HO-1 signaling pathway in this invention.

[0025] Figure 6 This is a graph showing the detection levels of genes and proteins related to oxidative stress damage mediated by the Nrf2 / HO-1 signaling pathway in this invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Example 1:

[0028] An embodiment of the present invention provides an m1A methyltransferase TRMT10C, the cDNA sequence of which is SEQ ID NO.1.

[0029] The expression and sequence of m1A methyltransferase TRMT10C were detected, as detailed below:

[0030] 1. Preparation of animal models of focal cerebral ischemia-reperfusion

[0031] A rat model of middle cerebral artery occlusion (MCAO) was established using a modified Longa suture occlusion method. Male SD rats aged 7-8 weeks, weighing 250-270g, were housed in an SPF-grade environment. First, the fur on the rat's neck was shaved to expose intact skin tissue. Then, a 1.5cm longitudinal incision was carefully made in the midline of the neck using surgical scissors. Simultaneously, a surgical microscope was turned on, and under the microscope, relatively large forceps were used to carefully dissect the tissues around the SD rat's neck to avoid mechanical damage. At this point, the muscles below the trachea were separated to expose the common carotid artery (CCA). Using microforceps, the muscles and fascia adhering to the CCA were further dissected, revealing the bifurcation of the CCA upwards, and further anteriorly, the internal carotid artery (ICA) and external carotid artery (ECA). The proximal end of the CCA near the bifurcation of the ECA was ligated. Since the suture could be deliberately avoided from entering the pterygopalatine artery (PPA), the PPA was not ligated. Then, a small incision was made in the vessel near the CCA ligation site using microscissors. A 0.18mm silicone-coated nylon suture was inserted into the CCA through this incision. To prevent massive arterial bleeding during insertion, a slipknot was tied at the midpoint of the vessel before insertion. The slipknot was then loosened and gently pushed, allowing the suture to advance along the course of the internal carotid artery. After passing the intersection of the ECA and ICA, it continued along the course of the ICA until it reached the middle cerebral artery (MCA) of the SD rat. The insertion was stopped when slight resistance was felt and further penetration was impossible. Calculated from the bifurcation, the suture had penetrated approximately 12-14mm into the MCA, essentially blocking the blood flow to the brain supplied by the middle cerebral artery of the SD rat. After 120 minutes of cerebral ischemia, the suture plug was slowly removed, the external carotid artery was ligated, and the suture at the common carotid artery was released to restore blood flow to the right carotid artery. The incision was sutured, disinfected with iodine, and the rats were placed in cages and housed at 25°C. The focal cerebral ischemia-reperfusion model was then established by MCAO. Throughout the procedure, local cerebral blood flow was monitored using a laser Doppler flowmeter to ensure successful model establishment. From the start of surgery until the animals regained consciousness, the body temperature of the SD rats was maintained at 36.5°C ± 1°C. In the sham-operated group, after anesthesia, only the bifurcation of the internal and external carotid arteries was exposed; no suture plug was inserted.

[0032] 2. Experimental grouping and drug administration

[0033] Male SD rats were weighed and randomly divided into 6 groups: sham operation group (Sham), cerebral ischemia model group (Vehicle), silent negative control group (sh-NC), TRMT10C silencing treatment group (sh-TRMT10C), overexpression negative control group (oe-NC), and TRMT10C overexpression intervention group (oe-TRMT10C).

[0034] The nucleic acid sequence of the shRNA is 5'-CCGTTGACAGTGGTTTCTGAA-3' (as shown in SEQ ID NO.2);

[0035] The nucleic acid sequence of the shRNA in the overexpression negative control group was 5'-TTCAGAAACCACTGTCAACGG-3' (as shown in SEQ ID NO.3) and served as the control.

[0036] Administration method: Based on rat body weight, anesthetize the rats with an intraperitoneal injection of 10% chloral hydrate (4.0 ml / kg), fix them in a prone position, and keep the skull horizontal and midline. Clip the hair, disinfect with povidone-iodine, expose the top of the skull, and wipe the top of the skull with an appropriate amount of H2O2 to expose the anterior fontanelle. According to the rat brain atlas, the coordinates of the left ventricle are: anteroposterior direction = 0.8 mm, mid-lateral direction = ±1.4 mm, dorsoventral direction = 3.5 mm relative to the bregma. Plasmids containing sh-TRMT10C or sh-NC, oe-TRMT10C or oe-NC are slowly injected into each lateral ventricle (3 μL per hemisphere, injection rate 0.15 μL / min, injection time exceeding 15 min), leaving the needle in place for 5 min after complete injection. After slowly raising and removing the micro-injection needle, seal the burr hole with bone wax. Remove the rat from the stereotaxic apparatus, suture the wound, disinfect with povidone-iodine, and return it to the rearing cage.

[0037] 3. Neuromotor function score

[0038] The modified neurological severity score (mNSS) was used to assess the neurological function of SD rats that underwent ischemia for 1 hour followed by reperfusion for 24 hours. The mNSS included five aspects: sensory testing, motor testing, balance beam testing, loss of reflexes and abnormal movements, epilepsy, myoclonus, and dystonia. The total score for this behavioral test was 18 points, with 0 points for normal rats and 18 points representing the highest possible behavioral deficit. The specific scoring for each item is as follows: mild impairment (1-6 points), moderate impairment (7-12 points), and severe impairment (13-18 points). Experimental results are as follows: Figure 1 As shown, the neuromotor score of the sh-TRMT10C silencing treatment group was significantly lower than that of the model group, and the neuromotor score of the sh-TRMT10C silencing treatment group was significantly lower than that of the shRNA-NC group. The results of the overexpression intervention group were exactly the opposite, indicating that silencing TRMT10C can significantly improve neuromotor dysfunction in SD rats after cerebral ischemia-reperfusion.

[0039] 4. Measurement of cerebral infarction volume

[0040] Twenty-four hours after ischemia-reperfusion, rats were decapitated and their brains harvested. Brain tissue was rapidly extracted, and the olfactory bulb, cerebellum, and lower brainstem were removed. The tissue was then frozen at -20°C for 20 minutes. Using a brain mold, the SD rat brain tissue was continuously sectioned coronally from 3 mm from the frontal pole, into five equally spaced sections. These sections were placed in 1% TTC and incubated at 37°C for 10 minutes, with the sections being turned occasionally to ensure uniform staining. After TTC staining, normal tissue appeared red, and infarcted tissue appeared white. ImageJ software was used to scan the SD rat brain slices, and the percentage of infarct volume relative to the contralateral brain volume was calculated. (See attached image.) Figure 2 As shown, compared with the model group and the negative control group, the silencing TRMT10C treatment group showed a significant reduction in cerebral infarction volume, while the overexpression intervention group showed the opposite result.

[0041] 5. HE staining

[0042] The sections were dried in a 65°C oven. Paraffin sections were dewaxed, typically with xylene or limonene, followed by hydration with a series of ethanol solutions. Hematoxylin was stained for 5-10 minutes; the stain penetrates the cell nucleus, giving it a blue-purple color. After a brief rinse, eosin was stained for 1-3 minutes, staining the cytoplasm and other tissue structures red. The stained sections required further dehydration with a series of ethanol solutions. Clearing was performed using xylene or other clearing agents. The sections were covered with mounting medium and then covered with coverslips. The stained tissue sections were observed and analyzed under an optical microscope to assess the degree and characteristics of brain injury. (See attached image) Figure 3 As shown, 24 hours after cerebral ischemia-reperfusion, compared with the sham-operated group, the number of cells in the cerebral cortex of rats in the model group and the negative control group was significantly reduced, the cell nuclei were condensed and the degree of vacuolization was severe; after TRMT10C silencing treatment, the number of cells in the cortex of SD rats was significantly increased compared with the model group, and the cell morphology was also restored. The results of the overexpression intervention group were exactly the opposite.

[0043] 6. TUNEL staining

[0044] Neuronal apoptosis was detected using a TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP terminal labeling) assay. Cell or brain tissue samples were fixed onto slides with ice-cold 4% glutaraldehyde for 10 minutes, followed by infiltration with 0.1% (v / v) Triton X-100 (Sigma) for 5 minutes. The slides were then incubated with the TUNEL incubation mixture at 37°C for 1.5 hours in a humid, dark environment. Cell nuclei were subsequently stained with 4,6-diphenylamine-2'-phenylhydrazine hydrochloride (DAPI). TUNEL-positive staining was observed and photographed using a fluorescence microscope. The apoptosis index is defined as the percentage of TUNEL-positive cells (positive cells / 100% total cells). (See attached image.) Figure 4As shown, 24 hours after cerebral ischemia-reperfusion, compared with the sham-operated group, the model group and the negative control group showed a significant increase in neuronal apoptosis in the cerebral cortex of rats; TRMT10C silencing treatment significantly reduced MCAO / R-induced neuronal apoptosis; the overexpression intervention group showed the opposite results.

[0045] 7. Real-time quantitative PCR (qRT-PCR) detection of NOX2, HO-1, and Nrf2 levels

[0046] Total RNA was extracted from tissues using the TRIzol method, and its concentration was determined by UV absorbance. Following the GenBank guidelines and instruction manual, RNA was reverse transcribed into cDNA using the SuperScript III First-Strand Synthesis kit with oligo(dT) primers. RT-PCR was then performed on 96-well plates using the SYBR Green PCR kit and an ABIPRIS M7500 real-time PCR system. RNA was reverse transcribed into cDNA, and PCR amplification was performed in 20 μL reaction volumes. The amplification conditions were: initial denaturation at 95°C for 30 seconds; denaturation at 95°C for 5 seconds; annealing at 60°C for 20 seconds; extension at 72°C for 20 seconds, for 40 cycles. The Ct values ​​of the samples were then obtained. Compared with GAPDH, 2... -ΔΔCT The relative mRNA expression levels of target genes were calculated. Compared with the sham-operated group, the mRNA expression level of the p53 signaling pathway in the cerebral cortex tissue of rats in the model group and the negative control group was increased; after TRMT10C silencing treatment in rats, the mRNA expression level of NOX2 was significantly decreased, and the mRNA expression level of the Nrf2 / HO-1 signaling pathway was significantly increased; the results in the overexpression intervention group were exactly the opposite. Figure 5 ).

[0047] 8. Western blotting (protein immunoblotting)

[0048] SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution, decapitated, and their brains were rapidly harvested on ice. The cerebral hemispheres were divided into the injured and uninjured sides. Fresh brain tissue from the peri-infarct area of ​​the injured side was collected. The brain tissue and lysis buffer were homogenized thoroughly at a mass-to-volume ratio of 1:10. The homogenate was placed in an ice box and thoroughly lysed on a shaker for 30 min. After centrifugation at 4°C and 12,000 rpm for 15 min, the supernatant was collected, and 6× protein loading buffer was added at a volume ratio. The mixture was denatured at 100°C for 15 min, aliquoted, and stored at -80°C. The protein samples were dissolved at -80°C, centrifuged, and added to the lanes using a microsyringe. SDS-polyacrylamide constant voltage gel electrophoresis was performed for separation, and the samples were transferred to PVDF membranes using an electroporation system. The membranes were blocked with 10% skim milk powder-TBST at room temperature for 1-2 h. Primary antibody was added and the membranes were incubated overnight at 4°C on a shaker. The next day, the primary antibody was discarded, and the membrane was washed with TBST for 10 min × 4 times. Horseradish peroxidase-labeled secondary antibody was added, and the membrane was incubated at room temperature on a shaker for 1 h. After washing with TBST, ECL luminescent substrate was added for color development. Analysis was performed using a Tanon 5200 fully automated chemiluminescence imaging system. The ratio of the target protein's gray value to the internal control GAPDH gray value was used for semi-quantitative analysis. Studies have shown that the expression levels of NOX2, HO-1, and Nrf2, which are related to oxidative stress injury signaling pathways, play a crucial role in cerebral ischemia-reperfusion injury. In oxidative stress injury caused by cerebral ischemia-reperfusion injury, NOX2 expression is significantly increased, and reducing its expression can significantly improve oxidative stress injury. The Nrf2 / HO-1 signaling pathway is upregulated in oxidative stress injury, and further activation of this pathway can also significantly improve oxidative stress injury. Figure 6 As shown, 24 hours after cerebral ischemia-reperfusion, compared with the sham-operated group, the protein expression levels of NOX2 and Nrf2 / HO-1 signaling pathways in the cerebral cortex tissue of SD rats in the model group and negative control group increased; after TRMT10C silencing treatment, the protein expression level of NOX2 was significantly reduced and the protein expression of Nrf2 / HO-1 signaling pathway was significantly upregulated; the results in the overexpression intervention group were exactly the opposite.

[0049] In summary, the present invention's downregulation of silent TRMT10C helps prevent cerebral ischemia / reperfusion injury and has a protective effect against acute neurological damage in SD rats with focal cerebral ischemia-reperfusion. It has promising applications in the preparation of drugs for treating ischemic stroke and provides a direction and technical foundation for finding and developing new, effective treatments for cerebrovascular diseases such as ischemic stroke, thus helping patients with these diseases to recover from their suffering as soon as possible.

[0050] Example 2:

[0051] A reagent for in vitro detection of m1A methyltransferase TRMT10C expression levels is provided in the preparation of a diagnostic kit for ischemic stroke. The reagent includes specific primers or probes targeting m1A methyltransferase TRMT10C. Those skilled in the art can readily design primers for amplifying the molecular marker or probes for identifying the molecular marker based on the molecular genetic markers according to the present invention, thereby for the diagnosis of ischemic stroke, for example, by PCR amplification using 2... -ΔΔCt The relative expression level of m1A methyltransferase TRMT10C was calculated using technical methods and compared with that of a healthy control group.

[0052] The above description is a detailed explanation of preferred feasible test examples of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. Application of m1A methyltransferase TRMT10C as a therapeutic target for stroke.

2. Application of inhibitors that inhibit the functional expression of m1A methyltransferase TRMT10C in the preparation of drugs for the treatment of stroke.

3. The application as described in claim 1 or 2, characterized in that, The nucleic acid sequence of the m1A methyltransferase TRMT10C is shown in SEQ ID NO.

1.

4. The application as described in claim 2, characterized in that, The inhibitor is an shRNA that targets and silences the expression of m1A methyltransferase TRMT10C; the nucleic acid sequence of the shRNA is shown in SEQ ID NO.

2.

5. A pharmaceutical composition, characterized in that, It contains an effective amount of an m1A methyltransferase TRMT10C inhibitor and a pharmaceutically acceptable carrier for the treatment of ischemic stroke.

6. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

7. The application of a reagent for in vitro detection of the expression level of m1A methyltransferase TRMT10C in the preparation of a diagnostic kit for ischemic stroke.

8. The application as described in claim 7, characterized in that, The reagents include specific primers or probes targeting the m1A methyltransferase TRMT10C.

Images