Application of synaptic binding protein 1 in preparation of product for preventing and / or treating ischemic heart disease

Abstract

The invention provides application of synaptic binding protein 1 in preparation of a product for preventing and / or treating ischemic heart disease, and belongs to the technical field of biological medicine. Researches find that overexpression of synaptic binding protein 1 can significantly inhibit myocardial infarction, myocardial cell necrosis and cardiac interstitial fibrosis of mice, reduce cardiac remodeling and improve cardiac functions, so that the purpose of effectively treating ischemic heart disease is achieved. By detecting the expression level of the synaptic binding protein 1, the risk of ischemic heart disease can be evaluated. The medicine for treating the ischemic heart disease provided by the invention is a heart disease treatment means, and provides a choice for clinical treatment of the ischemic heart disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an application of synaptotagmin 1 in the preparation of a product for preventing and / or treating ischemic heart disease. Background Art

[0002] Due to the limited proliferation capacity of mature cardiomyocytes, cardiac cell death is the root cause of heart disease. The commonly used clinical treatments for ischemic heart disease currently include drug therapy, coronary artery implantation surgery, surgical bypass surgery, etc., but drugs have potential adverse reactions, some drugs may even have an adverse effect on prognosis, and surgical treatment also has risks. Several forms of cell death with different morphologies have been found in damaged myocardium, including apoptosis, pyroptosis, necrosis, and autophagy. Necrosis is the main form of cell death in severely damaged hearts (such as ischemic hearts, ischemic / reperfusion injured hearts, and diabetic hearts). Therefore, inhibiting cell necrosis through anti-necroptosis therapy may be a way to improve patients. Therefore, finding molecules that inhibit myocardial necrosis has very important theoretical and clinical significance.

[0003] Synaptotagmin 1 (Syt1) is a Ca 2+ sensor, with 2 Ca 2+ Binding to the C2 domain (C2A and C2B) mediates synchronized neurotransmitter release. Initially, Syt1 has been shown to regulate endocytosis, vesicle trafficking, membrane repair, and synaptic plasticity during synaptic transmission. Syt1 and other members of the synaptotagmin family oligomerize into a ring-shaped vesicle clamp to promote vesicle docking and inhibit membrane fusion. Syt1 has been extensively and deeply implicated in the pathogenesis of neurodevelopmental disorders and alcohol addiction. Oxidative stress imbalance in gliomas is accompanied by downregulation of Syt1 mRNA. In diabetic encephalopathy, both the mRNA and protein levels of Syt1 are reduced during advanced glycation end products (AGE)-induced oxidative stress. However, Syt1 has not been developed as a drug target in cardiac ischemia-reperfusion (I / R) injury. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a use of synaptotagmin 1 in the preparation of a product for preventing and / or treating ischemic heart disease.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of synaptotagmin 1 in preparing a product for preventing and / or treating ischemic heart disease.

[0007] The present invention also provides an application of a synaptotagmin 1 overexpression reagent in the preparation of a product for preventing and / or treating ischemic heart disease.

[0008] The present invention also provides an application of a reagent for detecting the expression of synaptotagmin 1 in the preparation of a product for diagnosing the risk of ischemic heart disease.

[0009] Preferably, the ischemic heart disease includes one or more of myocardial infarction, cardiac interstitial fibrosis, cardiac remodeling and myocardial necrosis.

[0010] Preferably, the synaptotagmin 1 overexpression reagent comprises an adenovirus that overexpresses synaptotagmin 1.

[0011] Preferably, the reagent for detecting synaptotagmin 1 expression comprises one or more of a kit, a test strip, and a gene chip.

[0012] Preferably, the product comprises a reagent or a medicine.

[0013] The present invention also provides a method for screening drugs for treating ischemic heart disease, which includes the steps of using a test drug to treat a receptor that lowly expresses the above-mentioned synaptotagmin 1 and determining the expression level of synaptotagmin 1. When the expression level of synaptotagmin 1 in the receptor is significantly increased after the receptor is treated with the test drug, the test drug can be determined to be a drug for treating ischemic heart disease.

[0014] The present invention also provides a system for assessing the risk of suffering from ischemic heart disease, comprising a data processing device and a detection device, wherein the detection device is used to detect the expression level of synaptotagmin 1 of a sample to be tested and a healthy sample; the data processing device comprises a data input module, a data recording module, a data comparison module and a conclusion output module; the data input module is configured to input the expression level value of synaptotagmin 1 of the sample to be tested; the data recording module is configured to store the expression level value of synaptotagmin 1 of the sample to be tested and the expression level value of synaptotagmin 1 of the healthy sample; the data comparison module is configured to receive the expression level value of synaptotagmin 1 of the sample to be tested and the expression level value of synaptotagmin 1 of the healthy sample sent by the data input module, and compare the expression level value of synaptotagmin 1 of the healthy sample with the expression level value of synaptotagmin 1 of the sample to be tested from the data recording module; the conclusion output module is configured to receive the comparison result sent by the data comparison module, and judge the comparison result according to a predetermined judgment condition; the risk of suffering from ischemic heart disease of the sample to be tested is assessed.

[0015] The present invention also provides a drug for preventing and / or treating ischemic heart disease, which comprises the above-mentioned synaptotagmin 1 overexpression reagent and pharmaceutically acceptable excipients.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides an application of synaptotagmin 1 in the preparation of a product for preventing and / or treating ischemic heart disease. Studies have found that overexpression of synaptotagmin 1 can significantly inhibit myocardial infarction, myocardial cell necrosis, cardiac interstitial fibrosis, attenuate cardiac remodeling and improve cardiac function in mice, thereby achieving the purpose of effectively treating ischemic heart disease. By detecting the expression level of synaptotagmin 1, the risk of ischemic heart disease can be assessed. The drug for treating ischemic heart disease provided by the present invention is a new means of treating heart disease, and provides a new option for the clinical treatment of ischemic heart disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The protein level of Syt1 in the left ventricle was detected by Western blotting after I / R or sham operation in mouse hearts;

[0019] Figure 2 A is a graph showing the results of WB detection of Syt1 expression levels in Syt1 overexpressing adenovirus, negative control adenovirus and blank control group, and B is a statistical analysis graph showing the Syt1 expression levels in Syt1 overexpressing adenovirus, negative control adenovirus and blank control group;

[0020] Figure 3 After the hearts were injected with Syt1-overexpressing adenovirus or PBS buffer, I / R or sham surgery was performed, and the results of Evens blue / TTC staining were compared;

[0021] Figure 4 A shows the results of HE staining and analysis of fibrosis areas in different treatment groups; B shows the results of Masson trichrome staining and analysis of fibrosis areas in different treatment groups;

[0022] Figure 5 Results of TRITC-conjugated wheat germ agglutinin staining and analysis of the cross-sectional area of ​​cardiomyocytes in different treatment groups;

[0023] Figure 6 The echocardiographic LV fractional shortening (FS) results of different treatment groups;

[0024] Figure 7 The PI staining and statistical results of cardiomyocyte necrosis in different treatment groups, where red is PI staining, indicating necrotic cells; blue is DAPI staining, indicating cell nuclei; green is actin staining, indicating cytoskeleton;

[0025] Figure 8 Comparison results of lactate dehydrogenase (LDH) activity in different treatment groups. DETAILED DESCRIPTION

[0026] The present invention constructs an ischemic heart disease model through myocardial ischemia-reperfusion injury, and the sham operation group is used as a negative control, and then the expression level of synaptotagmin 1 (Syt1) is detected. The results show that the expression level of Syt1 is downregulated and has a significant expression difference. Therefore, Syt1 is used as a target for preventing and / or treating heart disease or for diagnosing or evaluating the risk of heart disease. Studies have shown that overexpression of synaptotagmin 1 can significantly inhibit myocardial infarction, myocardial cell necrosis, cardiac interstitial fibrosis, weaken cardiac remodeling and improve cardiac function in mice, thereby achieving the purpose of effectively treating ischemic heart disease.

[0027]

[0028] Based on this, the present invention provides an application of synaptotagmin 1 in the preparation of a product for preventing and / or treating ischemic heart disease.

[0029] The present invention also provides an application of a synaptotagmin 1 overexpression reagent in the preparation of a product for preventing and / or treating ischemic heart disease.

[0030] The present invention also provides an application of a reagent for detecting the expression of synaptotagmin 1 in the preparation of a product for diagnosing the risk of ischemic heart disease.

[0031] In the present invention, the ischemic heart disease includes one or more of myocardial infarction, cardiac interstitial fibrosis, cardiac remodeling and myocardial necrosis. The overexpression reagent of synaptotagmin 1 includes an adenovirus that overexpresses synaptotagmin 1. The construction method includes: cloning the synaptotagmin 1 coding sequence shown in SEQ ID NO.1 into the pShuttle-CMV adenovirus vector at the KpnI and EcoRV restriction sites to obtain a vector containing the synaptotagmin 1 coding sequence, i.e., the pShuttle-CMV-Syt1 adenovirus vector, mixing the pShuttle-CMV-Syt1 adenovirus vector and the pAdEasy-1 packaging vector to obtain a mixed solution; mixing RNAi-Mate with the mixed solution to obtain a transfection mixture, incubating the transfection mixture with HEK293A cells to obtain an adenovirus that overexpresses synaptotagmin 1.

[0032] In the present invention, the reagent for detecting the expression of synaptotagmin 1 includes one or more of a kit, a test strip, and a gene chip. The product includes a reagent or a drug.

[0033] The present invention also provides a method for screening drugs for treating ischemic heart disease, which includes the steps of using a test drug to treat a receptor that lowly expresses the above-mentioned synaptotagmin 1 and determining the expression level of synaptotagmin 1. When the expression level of synaptotagmin 1 in the receptor is significantly increased after the receptor is treated with the test drug, the test drug can be determined to be a drug for treating ischemic heart disease.

[0034] The present invention also provides a system for assessing the risk of suffering from ischemic heart disease, comprising a data processing device and a detection device, wherein the detection device is used to detect the expression level of synaptotagmin 1 of a sample to be tested and a healthy sample; the data processing device comprises a data input module, a data recording module, a data comparison module and a conclusion output module; the data input module is configured to input the expression level value of synaptotagmin 1 of the sample to be tested; the data recording module is configured to store the expression level value of synaptotagmin 1 of the sample to be tested and the expression level value of synaptotagmin 1 of the healthy sample; the data comparison module is configured to receive the expression level value of synaptotagmin 1 of the sample to be tested and the expression level value of synaptotagmin 1 of the healthy sample sent by the data input module, and compare the expression level value of synaptotagmin 1 of the healthy sample with the expression level value of synaptotagmin 1 of the sample to be tested from the data recording module; the conclusion output module is configured to receive the comparison result sent by the data comparison module, and judge the comparison result according to a predetermined judgment condition; the risk of suffering from ischemic heart disease of the sample to be tested is assessed.

[0035] In the present invention, the predetermined judgment condition is that the expression value of synaptotagmin 1 of the test sample is significantly downregulated compared with the expression value of synaptotagmin 1 of the healthy sample, indicating that the risk of ischemic heart disease in the test sample is high.

[0036] The present invention also provides a drug for preventing and / or treating ischemic heart disease, which comprises the above-mentioned synaptotagmin 1 overexpression reagent and pharmaceutically acceptable excipients.

[0037] In the present invention, the drug can use synaptotagmin 1 as the only active ingredient, or can be used in combination with other active drugs for preventing and / or treating ischemic heart disease to prevent and treat ischemic heart disease. The excipients include lentivirus, adenovirus and adeno-associated virus. The drug is administered orally or by injection; the injection administration method is preferably selected from intravenous injection, intramuscular injection, intracoronary injection and cardiac injection to achieve the purpose of preventing and / or treating ischemic heart disease. The ischemic heart disease includes one or more of myocardial infarction, cardiac interstitial fibrosis, cardiac remodeling and myocardial necrosis.

[0038] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] In the following examples, the Shuttle-CMV adenovirus vector was purchased from Addgene, catalog number #16403. The pAdEasy-1 packaging vector was purchased from Addgene, catalog number #16400.

[0041] Example 1

[0042] Detection of Syt1 expression in mice with ischemic heart disease

[0043] In this embodiment, mice were kept under pathogen-free conditions, the temperature of the breeding room was maintained at 21-26°C, the humidity was maintained at 50%-60%, and a 12-hour light and 12-hour dark cycle was provided. 7-week-old male C57BL / 6J mice were anesthetized with 2% isoflurane, the left anterior descending coronary artery (LAD) was ligated for 45 minutes, and then reperfused for 1 hour to construct a mouse model of myocardial ischemia-reperfusion injury (I / R group). The sham operation group (Sham group) served as the negative control group, in which the sham operation group did not ligate the left anterior descending coronary artery, and the rest of the operations were the same as those of the myocardial ischemia-reperfusion injury group. The chest was quickly opened, and the heart was flushed with normal saline. The hearts of each group were taken out, and after lysis with RIPA lysis buffer, the Syt1 protein in the heart tissue was extracted, and a protein immunoblotting experiment was performed to detect the expression level of Syt1.

[0044] If the expression level of Syt1 protein in mice with myocardial ischemia-reperfusion injury is significantly downregulated, it indicates that the mice are at high risk of developing heart disease.

[0045] Figure 1 The results showed that compared with the sham operation group, the expression level of Syt1 protein in the heart tissue of mice was significantly downregulated after myocardial ischemia-reperfusion injury. Therefore, the present invention can be used to determine the risk of heart disease by detecting the expression of Syt1.

[0046] Example 2 Detection of the role of Syt1 in ischemic heart disease

[0047] Construction of adenovirus overexpressing Syt1: Synthesize the coding sequence of Syt1 mRNA, as shown in SEQ ID NO.1, and clone the coding sequence of Syt1 mRNA into the pShuttle-CMV adenovirus vector at the KpnI and EcoRV restriction sites to obtain the pShuttle-CMV-Syt1 vector. Add 0.5 mL of serum-free DMEM to a sterile 1.5 mL centrifuge tube, then add the pShuttle-CMV-Syt1 vector and the pAdEasy-1 packaging vector, mix well, and obtain a mixed solution. Take another sterile 1.5 mL centrifuge tube, add 0.5 mL of serum-free DMEM, then add 10-15 μL RNAi-Mate, mix well, and place at room temperature for 5 minutes, then mix the two tubes, and place at room temperature for 20-25 minutes to obtain a transfection mixture. The transfection mixture was added dropwise to the HEK293A cell culture dish, and the culture dish was gently shaken back and forth to mix the complex, and incubated in a 37°C 5% CO2 incubator for 4 to 6 hours. The transfection solution was discarded, and 4 mL of DMEM culture solution containing 10% FBS was added, and the culture was continued at 37°C 5% CO2. When the cells appeared plaques (>50% of the cells remained), the virus lysate was collected to obtain the adenovirus overexpressing synaptotagmin 1.

[0048] Meanwhile, a negative adenovirus was constructed: the pShuttle-CMV-Syt1 adenovirus vector and the pAdEasy-1 packaging vector not connected to any target sequence were mixed to obtain a mixed solution; RNAi-Mate was mixed with the mixed solution to obtain a transfection mixture, and the transfection mixture was incubated with HEK293A cells to obtain a negative control adenovirus.

[0049] Seven-week-old male C57BL / 6J mice were anesthetized with 2% isoflurane, and 2 × 10 10 The adenovirus expressing Syt1 (Syt1 group) and negative control adenovirus (NC group) at moi were injected into the aortic root, pumped in the heart closed system for 20 seconds, and then the chest was closed and sutured. At the same time, the blank control group was not injected. After 48 hours, the total heart protein was extracted, and the expression level of Syt1 in the blank control group, overexpressing Syt1 adenovirus and negative control adenovirus was detected by western blot, and β-actin was used as an internal reference.

[0050] Figure 2 The results showed that the expression level of Syt1 in the Syt1 overexpressing adenovirus group was significantly increased compared with the negative control adenovirus group.

[0051] Seven-week-old male C57BL / 6J mice were anesthetized with 2% isoflurane and infected with the above-mentioned adenovirus expressing Syt1. After cross-clamping the aorta and pulmonary artery, 2×1010 The adenovirus expressing Syt1 (i.e., Ad-Syt1 group) and the negative control virus NC (WT group) described in moi were injected into the aortic root. Pumped for 20 seconds in a closed heart system. Five days later, the Ad-Syt1 group and the WT group mice were anesthetized again and the left anterior descending coronary artery (LAD) was ligated for 45 minutes, and then reperfused for 1 hour to obtain the Ad-Syt1-IR group and the WT-IR group. The sham operation group (Sham group) was used as a negative control group, in which the sham operation group was five days later, and the left anterior descending coronary artery was not ligated to the anesthetized Ad-Syt1 group and the WT group mice, and the Ad-Syt1-Sham group and the WT-Sham group were obtained. Then echocardiography and histological staining were performed respectively to evaluate the myocardial cell necrosis, myocardial infarction area, cardiac fibrosis and cardiac function.

[0052] The results showed that the infarct size assessed by evens blue / TTC staining was significantly reduced in Syt1-overexpressing mice after myocardial ischemia-reperfusion injury compared with the WT-IR group (see Figure 3 ).

[0053] Hematoxylin-eosin staining and Masson's trichrome staining showed that overexpression of Syt1 significantly inhibited cardiac interstitial fibrosis compared with the WT-IR group (see Figure 4 ).

[0054] TRITC-conjugated wheat germ agglutinin (WGA) staining and echocardiographic LV fractional shortening revealed that significantly attenuated cardiac remodeling and significantly improved cardiac function were observed in Syt1-overexpressing infarcted hearts compared with the WT-IR group ( Figure 5-6 ).

[0055] Propidium iodide (PI) staining and lactate dehydrogenase (LDH) activity assay showed that overexpression of Syt1 significantly inhibited myocardial necrosis compared with the WT-IR group (see Figure 7-Figure 8 ).

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of synaptotagmin 1 in the preparation of products for preventing and / or treating ischemic heart disease.

2. Use of a synaptotagmin 1 overexpression reagent in the preparation of a product for preventing and / or treating ischemic heart disease.

3. Application of reagents for detecting synaptotagmin 1 expression in the preparation of products for diagnosing the risk of ischemic heart disease.

4. The use according to any one of claims 1 to 3, characterized in that: The ischemic heart disease includes one or more of myocardial infarction, cardiac interstitial fibrosis, cardiac remodeling and myocardial necrosis.

5. The use according to claim 2, characterized in that: The reagent for overexpressing synaptotagmin 1 includes an adenovirus that overexpresses synaptotagmin 1.

6. The use according to claim 3, characterized in that: The reagent for detecting the expression of synaptotagmin 1 includes one or more of a kit, a test strip, and a gene chip.

7. The use according to any one of claims 1 to 3, characterized in that: The product includes a reagent or a drug.

8. A method for screening drugs for treating ischemic heart disease, characterized in that: The method comprises the steps of using a test drug to treat a receptor that under-expresses the synaptotagmin 1 of claim 1 or 2 and determining the expression level of synaptotagmin 1. When the expression level of synaptotagmin 1 in the receptor is significantly increased after the receptor is treated with the test drug, the test drug can be determined to be a drug for treating ischemic heart disease.

9. A system for assessing the risk of suffering from ischemic heart disease, characterized in that: It comprises a data processing device and a detection device, wherein the detection device is used to detect the expression amount of synaptotagmin 1 of the sample to be tested and the healthy sample; the data processing device comprises a data input module, a data recording module, a data comparison module and a conclusion output module; the data input module is configured to input the expression amount value of synaptotagmin 1 of the sample to be tested; the data recording module is configured to store the expression amount value of synaptotagmin 1 of the sample to be tested and the expression amount value of synaptotagmin 1 of the healthy sample; the data comparison module is configured to receive the expression amount value of synaptotagmin 1 of the sample to be tested and the expression amount value of synaptotagmin 1 of the healthy sample sent by the data input module, and compare the expression amount value of synaptotagmin 1 of the healthy sample with the expression amount value of synaptotagmin 1 of the sample to be tested from the data recording module; The conclusion output module is configured to receive the comparison result sent by the data comparison module and make a judgment on the comparison result according to a predetermined judgment condition; Assess the risk of ischemic heart disease in the sample to be tested.

10. A drug for preventing and / or treating ischemic heart disease, characterized in that: The drug comprises the synaptotagmin 1 overexpression agent according to claim 2 and pharmaceutically acceptable excipients.

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