Effect of TMEM159 in preparation of cerebral ischemia-reperfusion injury treatment medicine

Abstract

The invention discloses an effect of TMEM159 in preparation of a medicine for treating cerebral ischemia reperfusion injury. According to the application disclosed by the invention, molecular biology experiments find that the expression of the TMEM159 in cerebral ischemia reperfusion brain tissues is reduced, and single cell sequencing data in a public database shows that the TMEM159 is mainly expressed in neuronal cells; an AAV9-mediated nerve cell specific TMEM159 overexpression adeno-associated virus vector is further constructed to verify the application of TMEM159 as a treatment target, and the result shows that the TMEM159 overexpression can reduce the cerebral infarction area caused by cerebral ischemia and reduce the cell apoptosis level; therefore, the invention provides a new therapeutic target and medicine for the cerebral ischemia disease, and the TMEM159 overexpression adeno-associated virus provided by the invention has the advantages of long-term expression, low toxicity, low immunogenicity, high tissue specificity and the like, and has good application prospects and transformation values.

Description

Technical Field

[0001] The present invention relates to the role of TMEM159 in preparing a drug for treating cerebral ischemia-reperfusion injury, and belongs to the technical field of biomedicine. Background Art

[0002] Cerebral ischemia (e.g., ischemic stroke) is one of the leading causes of disability and mortality worldwide. Despite advances in thrombolytic therapy and intravascular intervention, the risk of cerebral reperfusion injury and the low clinical conversion rate of neuroprotective drugs still leave most patients facing irreversible neurological deficits. Among them, cerebral ischemia-reperfusion injury is a key issue that needs to be urgently addressed in the field of cerebral ischemia. Cerebral ischemia-reperfusion injury refers to a pathological process in which, when blood flow is restored to brain tissue after a brief period of ischemia, not only does brain function not improve, but tissue damage is further aggravated due to mechanisms such as the explosive generation of oxygen free radicals, intracellular calcium overload, mitochondrial dysfunction, and excessive inflammatory response. Its core mechanisms include: the massive accumulation of reactive oxygen species (ROS) after reperfusion triggers oxidative stress, damaging lipid, protein, and DNA structures; abnormal opening of the mitochondrial permeability transition pore (mPTP) leads to a collapse of cellular energy metabolism and activation of apoptotic signals; inflammasomes (such as NLRP3) trigger the release of proinflammatory cytokines (IL-1β, IL-18), recruiting immune cells to infiltrate and amplify tissue damage; at the same time, impaired integrity of the blood-brain barrier triggers brain edema and infiltration of peripheral immune cells. In addition, processes such as autophagy imbalance, endoplasmic reticulum stress, and pyroptosis are also involved. Therefore, exploring new molecular targets to develop therapeutic drugs for cerebral ischemia-reperfusion injury has become an urgent need in the field of cerebral ischemia research.

[0003] TMEM159 (Transmembrane Protein 159) is a multi-transmembrane protein located on the endoplasmic reticulum (ER) with a typical transmembrane domain structure. This protein is widely distributed in various tissues and participates in core biological processes regulating cellular homeostasis. Studies have shown that TMEM159 plays a key role in cellular stress responses by mediating protein interactions, including regulating ER-mitochondrial communication, influencing autophagosome formation and lysosomal degradation pathways, and participating in maintaining intracellular calcium homeostasis. Furthermore, TMEM159 can coordinate with the ubiquitin-proteasome system to regulate the stability of specific substrate proteins, thereby affecting cell cycle progression, apoptotic signaling, and metabolic reprogramming. Under pathological conditions, its expression level is closely correlated with inflammasome activation, oxidative stress tolerance, and regulation of the immune microenvironment, suggesting that it may participate in disease development through multidimensional molecular networks. However, the role of TMEM159 in cerebral ischemia-reperfusion injury remains unclear. Summary of the Invention

[0004] The present invention aims to address the shortcomings of existing technologies and provide a novel target for TMEM159 in cerebral ischemia-reperfusion injury. Cell and animal experiments have shown that overexpression of TMEM159 can alleviate neuronal apoptosis and reduce the size of cerebral infarction in mice.

[0005] To achieve the above objectives, the present invention provides the use of TMEM159 gene or protein as a drug target in the preparation or screening of drugs for preventing, improving and / or treating cerebral ischemia-reperfusion injury.

[0006] Preferably, the drug is a chemical drug or biological molecule that can increase the expression of TMEM159 gene and / or protein in hepatocytes in vivo, thereby preventing, improving and / or treating cerebral ischemia-reperfusion injury.

[0007] The present invention also provides a use of a TMEM159 gene or protein, or a promoter thereof, for preparing a composition or preparation for preventing, improving and / or treating cerebral ischemia-reperfusion injury.

[0008] Preferably, the TMEM159 promoter is selected from the group consisting of: a small molecule compound, a TMEM159 gene delivery vector, and a combination of one or more TMEM159 protein agonists.

[0009] Preferably, the gene delivery vector comprises a viral vector or a non-viral vector.

[0010] Preferably, the viral vector comprises an adeno-associated viral vector, and the non-viral vector comprises a liposome.

[0011] Preferably, the TMEM159 gene delivery vector is an adeno-associated viral vector, which uses a neuron-specific promoter to promote overexpression of the TMEM159 gene, and the vector backbone is AAV.

[0012] Preferably, the adeno-associated virus vector is an AAV.CAP-B10 serotype adeno-associated virus, and the AAV.CAP-B10 serotype adeno-associated virus is constructed by integrating the neuron-specific promoter Syn and the TMEM159 gene sequence and the reporter gene EGFP into the AAV.CAP-B10 vector skeleton to form a recombinant plasmid AAV.CAP-B10-Syn-TMEM159-EGFP, and the recombinant plasmid is then obtained through viral packaging and purification.

[0013] The present invention also provides a medicine or pharmaceutical composition for treating cerebral ischemia-reperfusion injury, which includes an active ingredient, wherein the active ingredient is an adeno-associated virus vector that overexpresses the TMEM159 gene, the adeno-associated virus vector uses a neuron-specific promoter to promote the overexpression of the TMEM159 gene, and the vector skeleton is AAV.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention found that TMEM159 expression was downregulated in brain tissues subjected to cerebral ischemia-reperfusion, and single-cell sequencing data in public databases showed that TMEM159 was mainly expressed in neuronal cells. In addition, AAV9-mediated neuronal cell-specific overexpression of TMEM159 can reduce the area of ​​cerebral infarction and the level of cell apoptosis. Adeno-associated virus (AAV) has become one of the most important gene vectors in the field of gene therapy due to its advantages such as long-term expression, low toxicity, low immunogenicity, and high tissue specificity. It makes up for the shortcomings of traditional cerebral ischemia-reperfusion therapy. Using adeno-associated virus to knock down TMEM159 to treat patients with cerebral ischemia-reperfusion injury can delay the progression of brain damage and improve the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : TMEM159 expression was downregulated in cerebral ischemia-reperfusion tissues; ***: P < 0.001.

[0017] Figure 2 : TMEM159 is mainly expressed in neuronal cells; A: Cluster diagram of Tmem159 expression in different cells of brain tissue; B: Violin plot and specific expression levels of Tmem159 expression in different cells of brain tissue.

[0018] Figure 3 : TMEM159 overexpression can reduce cerebral infarction caused by cerebral ischemia-reperfusion; ***: P < 0.001.

[0019] Figure 4 : Overexpression of TMEM159 can reduce apoptosis induced by cerebral ischemia-reperfusion injury; ***: P < 0.001. DETAILED DESCRIPTION

[0020] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0021] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in the following examples, unless otherwise specified, were obtained from commercial sources.

[0022] Example 1 Expression of TMEM159 in brain tissue after cerebral ischemia-reperfusion

[0023] In this example, RT-qPCR was used to detect the expression of TMEM159 in normal brain tissue and brain tissue subjected to cerebral ischemia-reperfusion. It was found that the expression of TMEM159 was downregulated in brain tissue subjected to cerebral ischemia-reperfusion. Further, single-cell sequencing data from public databases showed that TMEM159 was mainly expressed in neuronal cells, such as Figure 2 shown.

[0024] Example 2 Verification of the role of TMEM159 gene or protein in a cerebral ischemia-reperfusion mouse model

[0025] (I) Preparation of AAV9-mediated TMEM159 overexpression adeno-associated virus (AAV-TMEM159): In this example, the AAV9 serotype adeno-associated virus was injected into the tail vein to achieve peripheral delivery-mediated specific overexpression of TMEM159 in nerve cells. The neuron-specific promoter Syn and the TMEM159 gene sequence (NCBI GENE ID: 233806) and the reporter gene EGFP (for expression tracking) were constructed into the AAV.CAP-B10 vector backbone to form the recombinant plasmid AAV.CAP-B10-Syn-TMEM159-EGFP. The recombinant plasmid was co-transfected with the pHelper plasmid and the pAAV-R plasmid into AAV-293 cells, and the assembly of viral particles was completed using the intracellular environment. Cells and supernatants were collected 48-72 hours after transfection, and high-purity AAV particles were obtained by lysis, nuclease treatment, and gradient centrifugation or affinity chromatography purification. Finally, the genome titer was determined by qPCR, and the integrity of the capsid protein was detected by in vitro infection experiments or SDS-PAGE to ensure the infection efficiency and safety of the virus preparation. A single injection of 200 μL high-dose AAV9 virus suspension (containing 1×10 12 vg).

[0026] (II) Animal Model Construction and Experimental Grouping: C57BL / 6J mice weighing 18-26 g were anesthetized with isoflurane inhalation and continuously monitored to maintain body temperature at 36.5–37.5°C. During the surgical procedure, a skin incision was made along the midline of the neck, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated and exposed. After the ECA branches were ligated and severed, a mouse-specific MCAO suture (RWD) was inserted through the ECA stump to the ICA. The suture diameter was selected based on body weight (0.17-0.19 mm for mice weighing 18-21 g, 0.20-0.21 mm for mice weighing 21-25 g, and 0.22-0.23 mm for mice weighing 25-26 g). The suture was advanced to the origin of the middle cerebral artery (MCA) to occlude blood flow. During this time, a decrease in cerebral blood flow was verified in real time using laser speckle contrast imaging. After 2 hours of ischemia, the suture was slowly withdrawn to allow reperfusion. Neurological deficit scores were assessed 24 hours after surgery, and brain tissue was subsequently removed by decapitation for subsequent analysis. Mice were randomly divided into an AAV-CON+ modeling group and an AAV-TMEM159+ modeling group. AAV was injected into the tail vein after modeling to assess neurological deficit scores at the experimental endpoint. Brain tissue was then removed by decapitation for subsequent testing and analysis. The specific testing methods and analysis results are as follows:

[0027] 1) Detection of cerebral infarction area: TTC staining (2,3,5-triphenyltetrazolium chloride staining) was used to detect cerebral infarction area. After the animals were sacrificed, the brain was quickly removed and quickly frozen at -20°C for 20 minutes for slicing. 2mm thick brain slices were cut coronally and immersed in 1% TTC solution (incubated in the dark at 37°C for 15-30 minutes). After fixation with 4% paraformaldehyde, the infarcted area appeared white and the normal tissue appeared red. The proportion of infarct area was calculated using ImageJ and other software: infarct volume (%) = (contralateral hemisphere volume - healthy side same area volume) / contralateral hemisphere volume × 100%. The results showed that TMEM159 overexpression can reduce infarct area. Figure 3 shown.

[0028] 2) Detection of brain apoptosis: The following protocol was used to detect the expression of apoptosis-related proteins in brain tissue: Mice were sacrificed by cervical dislocation and brains were rapidly removed. The target brain region (e.g., the peri-infarct area) was isolated on ice, snap-frozen in liquid nitrogen, and stored at −80°C. Tissue blocks were then homogenized on ice with pre-chilled RIPA lysis buffer containing protease inhibitors at a 1:10 mass-to-volume ratio. The supernatant was collected by centrifugation at 4°C (12,000 rpm for 15 minutes). Protein concentration was determined by BCA assay and standardized, and the cells were denatured by boiling in SDS loading buffer. 20–40 μg of protein was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 hour and incubated sequentially with primary antibodies against cleaved caspase-3 (1:1000) and Bax / Bcl-2 (1:1000), followed by an HRP-conjugated secondary antibody (1:5000). After ECL development, the grayscale values ​​of the bands were analyzed using ImageJ (using β-actin as an internal reference). The entire experiment was performed on ice to avoid protein degradation, and a sham operation group and a normal control group were set up to ensure the specificity of the detected brain region and the reliability of the results. The results showed that TMEM159 overexpression can reduce the expression of apoptosis indicators, such as Figure 4 shown.

[0029] The above description is only a preferred embodiment of the present invention and does not constitute any formal or substantial limitation to the present invention. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of the TMEM159 gene or protein as a drug target in the preparation or screening of drugs for preventing, improving and / or treating cerebral ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that The drug is a chemical drug or biological molecule that can increase the expression of TMEM159 gene and / or protein in liver cells in vivo, thereby preventing, improving and / or treating cerebral ischemia-reperfusion injury.

3. A use of a TMEM159 gene or protein, or a promoter thereof, characterized in that: Used for preparing a composition or preparation for preventing, improving and / or treating cerebral ischemia-reperfusion injury.

4. The use according to claim 3, characterized in that The TMEM159 promoter is selected from the group consisting of: a small molecule compound, a TMEM159 gene delivery vector, and a TMEM159 protein agonist or a combination thereof.

5. The use according to claim 4, characterized in that The gene delivery vector includes a viral vector or a non-viral vector.

6. The use according to claim 5, characterized in that The viral vector includes an adeno-associated viral vector, and the non-viral vector includes a liposome.

7. The use according to claim 3, characterized in that The TMEM159 gene delivery vector is an adeno-associated viral vector, which uses a neuron-specific promoter to initiate overexpression of the TMEM159 gene, and the vector backbone is AAV.

8. The use according to claim 7, characterized in that The adeno-associated virus vector is an AAV.CAP-B10 serotype adeno-associated virus. The AAV.CAP-B10 serotype adeno-associated virus is constructed by integrating the neuron-specific promoter Syn and the TMEM159 gene sequence and the reporter gene EGFP into the AAV.CAP-B10 vector skeleton to form a recombinant plasmid AAV.CAP-B10-Syn-TMEM159-EGFP. The recombinant plasmid is then obtained through viral packaging and purification.

9. A medicine or pharmaceutical composition for treating cerebral ischemia-reperfusion injury, characterized in that: The invention comprises an active ingredient, wherein the active ingredient is an adeno-associated virus vector for overexpressing the TMEM159 gene. The adeno-associated virus vector uses a neuron-specific promoter to initiate overexpression of the TMEM159 gene, and the vector skeleton is AAV.

10. The drug or pharmaceutical composition according to claim 9, wherein The adeno-associated virus vector is an AAV.CAP-B10 serotype adeno-associated virus. The AAV.CAP-B10 serotype adeno-associated virus is constructed by integrating the neuron-specific promoter Syn and the TMEM159 gene sequence and the reporter gene EGFP into the AAV.CAP-B10 vector skeleton to form a recombinant plasmid AAV.CAP-B10-Syn-TMEM159-EGFP. The recombinant plasmid is then obtained through viral packaging and purification.

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