Application of USP18 gene as drug target in screening of drugs for preventing, relieving and / or treating myocardial ischemia-reperfusion injury

Abstract

The invention relates to application of a USP18 gene as a drug target in screening drugs for preventing, relieving and / or treating myocardial ischemia-reperfusion injury. Compared with a mouse without the USP18 gene knockout, the mouse with the USP18 gene knockout has the advantages that the myocardial infarction area is obviously reduced, the mitochondrial dysfunction is obviously improved, the cardiac contraction function is obviously enhanced, and the long-term myocardial fibrosis degree is reduced. The result shows that USP18 gene knockout can significantly alleviate the pathological process of myocardial ischemia reperfusion injury. Therefore, the USP18 gene is used as a target, and the screened USP18 gene expression inhibitor can be used for preparing medicines for preventing, relieving and / or treating myocardial ischemia-reperfusion injury. Compared with an Anti-NC mouse, the myocardial infarction area of the Anti-PTEN-L mouse is remarkably reduced, the mitochondrial dysfunction is remarkably improved, the cardiac contraction function is remarkably enhanced, and the long-term myocardial fibrosis degree is reduced. Therefore, the PTEN-L neutralizing antibody can also be used for preparing the medicine for preventing, relieving and / or treating the myocardial ischemia-reperfusion injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of USP18 gene as a drug target in screening drugs for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury. BACKGROUND

[0002] Acute myocardial infarction (AMI) is one of the cardiovascular diseases with the highest mortality worldwide, and its main treatment is to quickly restore coronary artery blood supply (such as percutaneous coronary intervention, PCI). However, myocardial ischemia-reperfusion (I / R) injury may be induced during perfusion, which is one of the major challenges in the treatment of AMI. I / R injury mainly manifests as oxidative stress, apoptosis, inflammatory response and myocardial remodeling, which may eventually lead to heart failure or sudden death.

[0003] In recent years, studies have shown that mitochondrial homeostasis plays a key role in regulating myocardial I / R injury. Mitophagy is an important mechanism for maintaining mitochondrial function by eliminating damaged mitochondria. The PINK1 / Parkin signaling pathway is the core pathway of mitophagy, and its activation can mark damaged mitochondria for autophagic degradation. The imbalance of mitophagy is considered to be an important cause of myocardial I / R injury. Therefore, targeting the regulatory mechanism of mitophagy has become an important direction for improving myocardial I / R injury.

[0004] However, it is not clear how to affect myocardial protection by regulating mitophagy, and further research is needed. SUMMARY

[0005] The present application provides a kind of USP18 gene as drug target in screening the application of the drug for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury, its screened USP18 gene expression inhibitor can be used to prepare the drug for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury.

[0006] In the first aspect, a kind of USP18 gene as drug target in screening the application of the drug for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury is provided.

[0007] In some embodiments, the drug includes a USP18 gene expression inhibitor, and the USP18 gene expression inhibitor includes a nucleic acid and / or an adenovirus.

[0008] In some embodiments, the nucleic acid includes siRNA, and the sequence of the siRNA is shown in SEQ ID NO.6. And / or, the adenovirus includes AD-shRNA or adeno-associated virus AAV-shRNA.

[0009] In a second aspect, there is provided a use of a USP18 gene expression inhibitor in the preparation of a medicament for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury.

[0010] In some embodiments, the USP18 gene expression inhibitor comprises a nucleic acid and / or an adenovirus.

[0011] In some embodiments, the nucleic acid comprises an siRNA, and the sequence of the siRNA is shown in SEQ ID NO. 6. In some embodiments, the adenovirus comprises an AD-shRNA or an adeno-associated virus (AAV)-shRNA.

[0012] In a third aspect, there is provided a use of a PTEN-L neutralizing antibody in the preparation of a medicament for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury.

[0013] In a fourth aspect, there is provided a medicament for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury, the medicament comprising one or more of a USP18 gene expression inhibitor and a PTEN-L neutralizing antibody.

[0014] In some embodiments, the USP18 gene expression inhibitor comprises a nucleic acid and / or an adenovirus.

[0015] In some embodiments, the nucleic acid comprises an siRNA, and the sequence of the siRNA is shown in SEQ ID NO. 6. In some embodiments, the adenovirus comprises an AD-shRNA or an adeno-associated virus (AAV)-shRNA.

[0016] The beneficial effects brought by the technical solutions provided in the present application include: The applicant found that, compared with mice without knockout of the USP18 gene, the myocardial infarction area of the USP18 gene knockout mice was significantly reduced, the mitochondrial dysfunction was significantly improved, the cardiac systolic function was significantly enhanced, and the degree of long-term myocardial fibrosis was reduced. These results show that the USP18 gene knockout can significantly reduce the pathological process of myocardial ischemia-reperfusion injury.

[0017] Based on the above findings, the present application finds a new use of the USP18 gene, i.e., the USP18 gene can be used as a target for screening a medicament for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury, and the USP18 gene expression inhibitor screened thereby can be used in the preparation of a medicament for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury.

[0018] Furthermore, the applicant also found that, compared with Anti-NC mice, Anti-PTEN-L mice showed significantly reduced myocardial infarction area, significantly improved mitochondrial dysfunction, significantly enhanced cardiac contractile function, and reduced long-term myocardial fibrosis. This indicates that PTEN-L neutralizing antibodies can also be used to prepare drugs for the prevention, mitigation, and / or treatment of myocardial ischemia-reperfusion injury. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The expression and statistical bar chart of USP18 protein in myocardial tissue of normal individuals and patients with ischemic cardiomyopathy provided in this application; Figure 2 The expression of USP18 protein in the myocardial tissue of mouse A provided in this application at 24 h, 72 h, and 4 weeks after sham surgery and ischemia-reperfusion surgery, and the statistical bar chart. Figure 3 The expression of USP18 protein in control and model cells of primary cultured neonatal rat cardiomyocytes provided in this application, and statistical bar charts. Figure 4 TTC staining images and infarct area statistics bar charts of the acute model groups of mice B and D provided in this application 24 h after ischemia-reperfusion. Figure 5 The images show cardiac slices obtained by electron microscopy from the acute model groups of mice B and D 24 h after ischemia-reperfusion, as provided in this application; the bar charts represent mitochondrial volume density and mitochondrial cristae loss (the left side of the bar chart is the acute model group of mice B, and the right side is the acute model group of mice D). Figure 6 Quantitative statistical analysis of H&E, PSR staining, cell size and left ventricular fibrosis area in mice in the B mouse chronic model group and D mouse chronic model group 4 weeks after ischemia-reperfusion provided in this application (the left side of the bar chart is the B mouse chronic model group, and the right side is the D mouse chronic model group). Figure 7 Statistical graphs showing the cardiac function test results of the B mouse chronic model group and the D mouse chronic model group before ischemia-reperfusion surgery and 2 weeks and 4 weeks after surgery, provided for this application. Figure 7 The numbers above represent p-values ​​compared to the chronic mouse model group (B mice). Figure 8TTC staining images and infarct area statistics bar charts of the neutralizing antibody treatment group and the control antibody treatment group of the acute model of mouse A provided in this application 24 h after ischemia-reperfusion (the left side of the bar chart is the control antibody treatment group of the acute model of mouse A, and the right side is the neutralizing antibody treatment group of the acute model of mouse A). Figure 9 The images show cardiac slices obtained by electron microscopy 24 h after ischemia-reperfusion in the A mouse acute model neutralizing antibody treatment group and the A mouse acute model control antibody treatment group provided in this application (the left bar chart is the A mouse acute model control antibody treatment group, and the right bar chart is the A mouse acute model neutralizing antibody treatment group). Figure 10 The statistical data of H&E and cell size of mice in the A mouse chronic model neutralizing antibody treatment group and the A mouse chronic model control antibody treatment group after 4 weeks of ischemia-reperfusion provided in this application are shown in the bar chart (the left side of the bar chart is the A mouse chronic model control antibody treatment group, and the right side is the A mouse chronic model neutralizing antibody treatment group). Figure 11 The bar charts provided in this application show the statistical results of cardiac function tests in the A mouse chronic model neutralizing antibody treatment group and the A mouse chronic model control antibody treatment group before ischemia-reperfusion surgery, and at 2 weeks and 4 weeks after surgery (the left side of the bar chart is the A mouse chronic model control antibody treatment group, and the right side is the A mouse chronic model neutralizing antibody treatment group). Figure 11 The numbers above represent p-values ​​compared to the antibody treatment group (a chronic mouse model control group). Figure 12 The following are the verification and statistical graphs of USP18 protein expression after transfection with NC-siRNA and USP18-siRNA provided for this application; Figure 13 The results of Caspase-3 enzyme activity detection for the NC-siRNA control group cells and USP18-siRNA control group cells, as well as the NC-siRNA control group model cells and USP18-siRNA control group model cells provided in this application. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0022] Ubiquitination and deubiquitination play important roles in cellular homeostasis, and ubiquitin-specific protease 18 (USP18) is a deubiquitinating enzyme.

[0023] This application used cardiomyocyte-specific USP18 gene knockout mice (denoted as USP18fl / fl / α-MHC-MerCreMer) and littermate wild-type mice with non-knockout USP18 gene (denoted as USP18fl / fl) as experimental subjects. A myocardial injury animal model was induced by ischemia-reperfusion (I / R) surgery to investigate the function of the USP18 gene. The results showed that USP18 expression was significantly upregulated in the myocardial ischemia-reperfusion injury model compared to the normal group. Compared with littermate wild-type USP18fl / fl model mice, the USP18fl / fl / α-MHC-MerCreMer model mice showed significantly smaller myocardial infarction area, significantly improved mitochondrial dysfunction, significantly enhanced cardiac contractile function, and reduced fibrosis. These results indicate that cardiomyocyte-specific USP18 gene knockout can significantly alleviate the pathological process of myocardial ischemia-reperfusion injury.

[0024] Further research revealed that USP18 exacerbates myocardial ischemia-reperfusion injury by deubiquitinizing and stabilizing PTEN-L, thereby inhibiting PINK1 / Parkin-mediated mitophagy. Treatment with PTEN-L neutralizing antibodies significantly reduced myocardial ischemia-reperfusion injury, improved mitochondrial dysfunction, reduced infarct size, and improved cardiac function and the degree of cardiac fibrosis. This indicates that PTEN-L not only participates in myocardial injury as a downstream effector molecule of USP18, but also that intervention with neutralizing antibodies can effectively reduce myocardial ischemia-reperfusion injury.

[0025] Therefore, the USP18 gene and its downstream molecule PTEN-L can serve as drug targets. In vitro cell or animal models with USP18 or PTEN-L gene knockout can be constructed to screen for drugs and / or biological agents that prevent, alleviate, and / or treat myocardial ischemia-reperfusion injury. These models can then be used through genetic engineering or neutralizing antibody therapy to achieve the same goal. Furthermore, small molecule inhibitors can be designed targeting USP18 or PTEN-L. In vitro cell or animal models with USP18 or PTEN-L gene overexpression can be used to screen for molecules that specifically inhibit USP18 or PTEN-L, thus providing new therapeutic molecules for the treatment of myocardial ischemia-reperfusion injury.

[0026] Based on the above findings, this application provides the use of the USP18 gene as a drug target in screening drugs for the prevention, mitigation and / or treatment of myocardial ischemia-reperfusion injury.

[0027] The drug in question is one that inhibits the expression of the USP18 gene.

[0028] Specifically, the drug includes a USP18 gene expression inhibitor.

[0029] Furthermore, USP18 gene expression inhibitors are substances that inhibit the expression of the USP18 gene, prepared or screened with the USP18 gene as the target.

[0030] The USP18 gene expression inhibitors include nucleic acids and / or adenoviruses.

[0031] The nucleic acid includes siRNA, the sequence of which is shown in SEQ ID NO.6.

[0032] The adenovirus includes AD-shRNA or adeno-associated virus AAV-shRNA.

[0033] siRNA can exert its effects through transfection, specifically by entering cardiomyocytes and reducing USP18 expression.

[0034] Based on the above findings, this application also provides the use of USP18 gene expression inhibitors in the preparation of drugs for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury.

[0035] Based on the above findings, this application also provides the use of PTEN-L neutralizing antibodies in the preparation of medicaments for the prevention, mitigation and / or treatment of myocardial ischemia-reperfusion injury.

[0036] PTEN-L neutralizing antibodies can be administered via injection.

[0037] Based on the above findings, this application also provides drugs for preventing, alleviating and / or treating myocardial ischemia-reperfusion injury.

[0038] The drug includes a USP18 gene expression inhibitor.

[0039] Alternatively, the drug may include a PTEN-L neutralizing antibody.

[0040] Alternatively, the drug may be a composition comprising a USP18 gene expression inhibitor and a PTEN-L neutralizing antibody. The USP18 gene expression inhibitor may be one or more of these inhibitors.

[0041] The PTEN-L neutralizing antibody specifically binds to the PTEN protein, inhibiting the interaction between the PTEN protein and USP18.

[0042] The above-mentioned compositions are in, but are not limited to, the following two forms: 1. Independent formulations: USP18 gene expression inhibitors and other drugs for the prevention, mitigation and / or treatment of myocardial ischemia-reperfusion injury are formulated as independent formulations.

[0043] 2. Compound preparations: USP18 gene expression inhibitors and other drugs for the prevention, relief and / or treatment of myocardial ischemia-reperfusion injury are formulated into compound preparations.

[0044] The present application is further illustrated below through examples.

[0045] Laboratory animals and their care: For ease of understanding, the mouse names used in the following experiments are defined as follows: C57BL / 6J mice: denoted as A mice.

[0046] USP18fl / fl mice: referred to as B mice.

[0047] α-MHC-MerCreMer mice: referred to as C mice.

[0048] USP18fl / fl / α-MHC-MerCreMer mice: designated as D mice, and D mice are obtained by mating B mice with C mice.

[0049] Experimental animals: 8-10 week old C57BL / 6J mice (i.e., A mice), α-MHC-MerCreMer mice (i.e., C mice), USP18fl / fl / α-MHC-MerCreMer mice (i.e., D mice) and littermate wild-type USP18fl / fl mice (i.e., B mice) with a weight of 25.5±2.0g were selected as experimental subjects.

[0050] Housing environment: All laboratory mice were housed in a specific pathogen-free (SPF) laboratory animal center.

[0051] Feeding conditions: The room temperature should be between 22-24℃, the humidity between 50-70%, the light should be alternating between light and dark for 12 hours, and the animals should have free access to water and food.

[0052] Example 1 Construction of cardiomyocyte-specific USP18 gene knockout (USP18fl / fl / α-MHC-MerCreMer) mice, i.e., construction of D mice.

[0053] Eight- to ten-week-old B mice (from the Southern Model Animal Center) were mated with C mice (purchased from the Jackson Laboratory, USA). After mating, the mice's tails were clipped for enzymatic gene identification, and D mice were obtained through screening. The primers used for the enzymatic method are as follows: F1 (sequence of SEQ ID NO.1): 5'-GGGGAGAGTTTACAGGTGGC-3'; R1 (sequence of SEQ ID NO.2): 5'-GGTTCCCAGAGCCAAGAGTC-3'; F1 (sequence of SEQ ID NO.3): 5'-GTGTTGATGCTAGGTGGGCA-3'; R1 (sequence is SEQ ID NO.4): 5'-GCCCAGCTGTTTCAGGTTTC-3'.

[0054] Example 2 Expression of USP18 in normal individuals and ischemic cardiomyopathy.

[0055] Hearts from healthy individuals and patients with ischemic cardiomyopathy were selected. Proteins were extracted from the hearts and subjected to SDS-PAGE-Western blot assay. The USP18 antibody (purchased from Cell Signaling Technology, #4813) specifically identified the USP18 protein, and β-actin was used as an internal control.

[0056] Test results as follows Figure 1 As shown, compared with normal people (i.e. Figure 1 Compared to the myocardial tissue of patients with ischemic cardiomyopathy (i.e., those with myocardial tissue in the middle stage), the myocardial tissue of patients with ischemic cardiomyopathy (i.e., those with myocardial tissue in the middle stage) is significantly different. Figure 1 USP18 protein expression was significantly upregulated in myocardial tissue of patients with ischemic cardiomyopathy (IHD). The expression level of USP18 protein in normal myocardial tissue was significantly different from that in patients with ischemic cardiomyopathy (P=0.0002).

[0057] Example 3 Myocardial ischemia-reperfusion model was established using mice A, B, and D. The resulting mice were designated as mouse model group A, mouse model group B, and mouse model group D, respectively.

[0058] Based on the reperfusion time, the mice were divided into acute and chronic models. The acute model was defined as mice reperfused for 24 hours, and these mice were designated as the A mouse acute model group, the B mouse acute model group, and the D mouse acute model group. The chronic model was defined as mice reperfused for 4 weeks, and these mice were designated as the A mouse chronic model group, the B mouse chronic model group, and the D mouse chronic model group.

[0059] The specific steps are as follows: Preoperative preparation: Before establishing the animal model of myocardial ischemia-reperfusion injury, 8-10 week old male A, B, and D mice were anesthetized and intraperitoneally injected with 3% sodium pentobarbital (40 mg / kg) to ensure the mice were pain-free during the operation. Mice were housed in a specific pathogen-free (SPF) environment with controlled temperature and humidity, free access to food and water, and a 12-hour light / 12-hour dark cycle to ensure they were in good physiological condition before surgery.

[0060] Intraoperative procedures: During the surgery, a left thoracotomy was first performed on the mice to expose the heart. The left anterior descending artery (LAD) was ligated using 7-0 silk suture 1 mm below the apex of the left atrial appendage. A PE-10 catheter was placed on the artery during ligation to prevent complete occlusion. Ischemia was confirmed by ST-segment elevation and localized cardiac pallor on an electrocardiogram (ECG). Forty-five minutes after ligation, the suture was released, the PE-10 catheter was removed, and blood flow was restored, thus achieving reperfusion. The entire procedure was performed under sterile conditions to minimize the risk of infection.

[0061] Postoperative care: Mice were administered butorphanol (Temgesic, 0.1 mg / kg) once daily for 6 days postoperatively to relieve pain. Serum and cardiac tissue samples were collected at specified time points after reperfusion (e.g., 24 hours or 4 weeks) for subsequent analysis.

[0062] In addition, sham surgery was performed: mice A, B, and D underwent sham surgery, and the mice after sham surgery were designated as the A mouse sham surgery group, B mouse sham surgery group, and D mouse sham surgery group, respectively. The left anterior descending artery (LAD) of the heart was not ligated in the sham-operated mice, and the remaining steps were the same as those described above for constructing the animal model of myocardial ischemia-reperfusion injury.

[0063] Example 4: Proteins were extracted from the hearts of mice in the sham-operated group and the mouse model group and subjected to SDS-PAGE-Western blot assay. The USP18 antibody (purchased from Cell Signaling Technology, #4813) specifically recognized the USP18 protein, and β-actin was used as an internal control.

[0064] Test results as follows Figure 2As shown, 0h represents sham surgery. The levels of USP18 protein in myocardial tissue in the sham surgery group were significantly higher than those at 24h, 72h, and 4w post-reperfusion (P<0.0001). Compared with the sham surgery group in mouse A, the expression of USP18 protein in myocardial tissue was significantly upregulated in the mouse A model group, and this upregulation gradually increased with the duration of reperfusion (24h, 72h, 4w).

[0065] Example 5: The A mouse model group was treated with both control antibody and PTEN-L neutralizing antibody. To inhibit PTEN-L, the A mouse model group was administered PTEN-L neutralizing antibody (Merck Millipore, MABS1680) at a dose of 50 μg per mouse every other day for 28 consecutive days following cardiac I / R surgery. These mice were designated as the A mouse model neutralizing antibody treatment group. The control group was set up by receiving the same solvent and IgG antibody (Sigma) from the A mouse model group; these mice were designated as the A mouse model control antibody treatment group.

[0066] As in Example 3, the A mouse model group can be divided into the A mouse acute model group and the A mouse chronic model group. Therefore, after the treatment in Example 5, the A mouse model neutralizing antibody treatment group can also be divided into the A mouse acute model neutralizing antibody treatment group and the A mouse chronic model neutralizing antibody treatment group. The A mouse model control antibody treatment group can also be divided into the A mouse acute model control antibody treatment group and the A mouse chronic model control antibody treatment group.

[0067] Example 6 Mouse heart TTC staining After modeling, cardiac tissue from both the B and D mouse acute model groups was collected for TTC staining to calculate the infarct area. The mice in both groups were sacrificed 24 hours after reperfusion, and their hearts were removed and frozen at -20°C for 20 minutes. Subsequently, the hearts were sectioned into 4-5 μm thick slices. The slices were stained with 1% TTC solution at 37°C for 15 minutes to distinguish between the infarcted area (IA, appearing pale) and the non-infarcted area (appearing red). The infarct area (IA) and total left ventricular area (LV) were quantified using Image-Pro Plus 6.0 software, and the percentage of infarct area to total left ventricular area (IA / LV) was calculated. For ease of presentation, the percentage IA / LV is represented as %LV in the graph.

[0068] like Figure 4As shown, the myocardial infarction area in the acute model group of mice B was significantly reduced compared to that in the acute model group of mice D (P=0.0001). For ease of understanding, WT represents the type associated with mice B, and KO represents the type associated with mice D. Figure 4 In the figure, WT represents the acute model group of B mice, and KO represents the acute model group of D mice.

[0069] Similarly, TTC staining was performed on the neutralizing antibody treatment group and the control antibody treatment group in the acute A mouse model 24 hours after ischemia-reperfusion. Figure 8 As shown, the myocardial infarction area of ​​the control antibody treatment group in the acute model of mouse A was significantly smaller than that of the control antibody treatment group in the acute model of mouse A (i.e., Anti-PTEN-L in the figure) than that of the control antibody treatment group in the acute model of mouse A (i.e., Anti-NC in the figure) (P=0.0002).

[0070] Example 7 Mitochondrial morphology was observed in sections of mice from the acute model groups B and D using electron microscopy.

[0071] First, take a 1mm sample from the apex of the left ventricle. 3 Heart tissue was fixed in 2.5% glutaraldehyde solution. It was then dehydrated using a gradient of ethanol and embedded in epoxy resin. After embedding, the tissue was cut into ultrathin sections of 60-90 nm using an ultramicrotome. The sections were double-stained with uranium acetate and lead citrate, and then observed and photographed using a Tecnai-20 transmission electron microscope (Philips-FEI). Quantitative analysis of mitochondrial volume density and cristae loss was performed using ImageJ software. 15-18 images were analyzed from each sample group to assess changes in mitochondrial morphology and number.

[0072] like Figure 5 As shown, the mitochondrial volume density in the myocardial tissue of the B mouse acute model group was significantly lower than that of the D mouse acute model group (P=0.0003); the mitochondrial cristae loss in the myocardial tissue of the B mouse acute model group was significantly lower than that of the D mouse acute model group (P=0.0001). Compared with the B mouse acute model group, the D mouse acute model group showed increased mitochondrial volume density and decreased cristae loss, indicating improved mitochondrial function. Figure 5 In the figure, WT represents the acute model group of B mice, and KO represents the acute model group of D mice.

[0073] Similarly, mitochondrial morphology was observed by electron microscopy in both the neutralizing antibody treatment group and the control antibody treatment group in the acute A mouse model 24 hours after ischemia-reperfusion. Figure 9 As shown, the mitochondrial volume density in the myocardial tissue of the control antibody treatment group in the acute mouse model A was significantly lower than that in the neutralizing antibody treatment group in the acute mouse model A (P=0.0001); the mitochondrial cristae loss in the myocardial tissue of the control antibody treatment group in the acute mouse model A was significantly lower than that in the neutralizing antibody treatment group in the acute mouse model A (P=0.0014). Compared with the control antibody treatment group in the acute mouse model A (i.e., Anti-NC in the figure), the neutralizing antibody treatment group in the acute mouse model A (i.e., Anti-PTEN-L in the figure) showed increased mitochondrial volume density and decreased mitochondrial cristae loss.

[0074] Example 8 Pathological examination Pathological examinations were performed on the chronic model groups of mice B and D, including H&E staining and PSR staining, to determine cardiomyocyte size and degree of fibrosis.

[0075] Embedding: Heart tissues from the chronic model groups of mice B and D were collected 4 weeks after surgery, fixed in 4% neutral formaldehyde solution for 48 hours, then dehydrated and embedded in paraffin. The section thickness was 5 μm.

[0076] H&E staining: After dewaxing and hydration, the sections were stained with hematoxylin solution for 10 minutes, followed by eosin solution for 5 minutes. After staining, the sections were dehydrated, cleared, and mounted. The sections were then observed and images were taken under a microscope.

[0077] PSR staining: The sections were mounted on glass slides, dewaxed and hydrated, pre-stained with picric acid solution for 5 minutes, and then stained with Sirius red solution for 30 minutes. After staining, the sections were rapidly washed, differentiated, dehydrated, cleared, and mounted. The sections were then observed and images were taken under a microscope.

[0078] H&E staining was used to assess cardiomyocyte morphology and cross-sectional area, and PSR staining was used to assess collagen deposition and fibrosis area. Following H&E staining, the cross-sectional area of ​​cardiomyocytes was measured using Image-Pro Plus 6.0 software; more than 200 cells per group were analyzed from 5 mice. Following PSR staining, the same software was used to quantify collagen deposition area and calculate the percentage of left ventricular fibrosis area relative to the total left ventricular area. These steps were used to assess cardiac structural changes and the degree of fibrosis following myocardial ischemia-reperfusion injury.

[0079] likeFigure 6 As shown, the size of cardiomyocytes in the chronic model group B was significantly different from that in the chronic model group D (P=0.0002); the degree of myocardial fibrosis in the chronic model group B was also significantly different from that in the chronic model group D (P=0.0002). Compared with the chronic model group B, the cardiomyocytes in the chronic model group D were smaller and the degree of fibrosis was lower. Figure 6 In the table, WT represents the chronic model group of B mice, and KO represents the chronic model group of D mice.

[0080] Similarly, in the chronic A mouse model, both the antibody-treated group and the control antibody-treated group underwent pathological H&E staining 4 weeks after modeling, but PSR staining was not performed. Figure 10 As shown, comparing the cardiomyocyte size of the control antibody treatment group and the neutralizing antibody treatment group of the chronic A mouse model, P=0.0012, the cardiomyocytes of the mice in the neutralizing antibody treatment group of the chronic A mouse model (i.e., Anti-NC in the figure) were smaller than those in the control antibody treatment group (i.e., Anti-PTEN-L in the figure).

[0081] Example 9 Mouse cardiac ultrasound Cardiac function was assessed by echocardiography in both the B mouse chronic model group and the D mouse chronic model group before ischemia-reperfusion surgery and at 2 and 4 weeks post-surgery. The testing steps are as follows: Mice were anesthetized with 1.5-2% isoflurane and evaluated using echocardiography on a MyLab 30CV system (Biosound Esaote, Inc.) equipped with a 15 MHz probe. Evaluation time points included preoperatively, 2 weeks postoperatively, and 4 weeks postoperatively. Measured parameters included left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVISd), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and heart rate (HR).

[0082] like Figure 7 As shown in Table 1 below, compared with the B mouse chronic model group, the D mouse chronic model group showed improved cardiac function. Figure 7 In the table, WT represents the chronic model group of B mice, and KO represents the chronic model group of D mice. For ease of understanding,Figure 7 "0w" in the text indicates preoperative.

[0083] Table 1

[0084] Echocardiography was performed 4 weeks after reperfusion in both the antibody-treated group and the control antibody-treated group of the chronic A mouse model. (See [link to original text]). Figure 11 As shown in Table 2 below, compared with the control antibody treatment group in the chronic A mouse model, the cardiac function in the neutralizing antibody treatment group in the chronic A mouse model was significantly improved.

[0085] Table 2

[0086] Example 10 Culture of primary cardiomyocytes from neonatal rats and construction of a cellular hypoxia-reoxygenation model: Culture of primary cardiomyocytes from neonatal rat suckling mice (1) Take 10 SD rat pups aged 1-3 days, disinfect the pups with 75% alcohol except for the head, remove the heart with ophthalmic scissors and micro forceps, and place the removed heart in a glass culture dish containing 10 mL of DMEM / F12 medium. Repeat the same procedure for the remaining pups.

[0087] (2) The heart was washed with DMEM / F12 medium and the heart tissue was cut into pieces with a volume of 1-2 mm. 3 Small pieces of heart tissue were cut and transferred to a serum bottle containing a stir bar. The DMEM / F12 medium was removed, and trypsin digestion solution was added. The tissue was digested on a shaker at 37°C for 15 minutes, allowed to stand for a short time, and then the supernatant was discarded.

[0088] (3) Add trypsin digestion solution again, mix well by pipetting, collect the supernatant after the tissue block precipitates, and terminate the digestion process with fetal bovine serum. Then store the mixture in a refrigerator at 4°C. Repeat this digestion step several times until the tissue block turns white and shrinks significantly. When collecting the supernatant, it should be done as thoroughly as possible to ensure that all cells are collected.

[0089] (4) Centrifuge the collected cardiomyocyte suspension at 1500 rpm for 8 minutes, and discard the supernatant after centrifugation. Add an appropriate amount of culture medium to the centrifuge tube, gently pipette to resuspend the cells, and concentrate the cell suspension into a 50 mL centrifuge tube. Filter the cell suspension through a 40 μm cell filter to remove undissociated tissue fragments.

[0090] (5) The filtered cell suspension was seeded into 100 mm culture dishes and allowed to adhere for 90 minutes. Subsequently, the unadhered cell suspension was aspirated and filtered again through a cell filter. After centrifugation and resuspending, the cells were counted, and BrdU (5-bromodeoxyuridine, final concentration 0.1 mM) was added. After mixing thoroughly, the cell suspension was seeded into culture dishes pre-coated with 0.1% gelatin.

[0091] (6) Gently shake the culture dish to disperse the cells and avoid creating vortices. Incubate the cells in a 37°C, 5% CO2 incubator for 48 hours. After incubation, wash the cells once with PBS and replace with fresh culture medium.

[0092] Modeling hypoxia-reoxygenation in neonatal rat primary cardiomyocytes: Hypoxia treatment: Cardiomyocytes were incubated in hypoxia buffer at 95% N2 and 5% CO2 for 4 hours.

[0093] Reoxygenation treatment: After hypoxia treatment, cells were incubated overnight (0h, 6h, 12h, 24h) in DMEM / F12 medium containing 10% fetal bovine serum (FBS) under normal culture conditions (95% air and 5% CO2).

[0094] For ease of understanding, primary cardiomyocytes from neonatal rats are referred to as control group cells. Cells from neonatal rats that have undergone hypoxia-reoxygenation modeling treatment are referred to as model group cells.

[0095] Proteins were extracted from control and model cells and subjected to SDS-PAGE-Western blot assays. The USP18 antibody (Cell Signaling Technology, #4813) specifically binds to the USP18 protein, with β-actin used as an internal control. Results are as follows: Figure 3 As shown, the USP18 protein content in the control group cells was significantly different from that in the model group cells after 4 hours of hypoxia followed by 6 hours of reoxygenation (P=0.0018); compared with the model group cells after 4 hours of hypoxia followed by 12 hours of reoxygenation (P<0.0001); and compared with the model group cells after 4 hours of hypoxia followed by 24 hours of reoxygenation (P<0.0001). The expression of USP18 protein in the model group cells increased and was upregulated with prolonged reoxygenation time. Figure 3 In the table, 0h represents the control group cells, and 6h, 12h, and 24h represent the model group cells after the corresponding time of hypoxia-reoxygenation modeling.

[0096] Example 11 siRNA transfection siRNA design: A specific siRNA was designed targeting the USP18 gene, while a control group (NC-siRNA) was set up using a non-targeted scrambled siRNA. The designed sequence is as follows.

[0097] NC-siRNA: 5′-GGAGAUGAAUCUAGAAGAAdTdT-3′ (SEQ ID NO.5); USP18-siRNA: 5′-UUCUUCUAGAUUCAUCUCCdTdT-3′ (SEQ ID NO. 6).

[0098] Transfection process: (1) Culture primary cardiomyocytes of newborn rats (i.e., control group cells) to an appropriate density (usually 70-80% confluence).

[0099] (2) Following the instructions of the Lipo 6000™ kit, mix the siRNA with the transfection reagent to form a transfection complex.

[0100] (3) Add the transfection complex to the culture dish and incubate with the cells for 4-6 hours.

[0101] (4) After incubation, replace with fresh culture medium and continue culturing the cells for subsequent experiments.

[0102] Among them, the control group cells were transfected with NC-siRNA to obtain NC-siRNA control group cells, and the control group cells were transfected with USP18-siRNA to obtain USP18-siRNA control group cells.

[0103] Verify mRNA knockdown efficiency Proteins were extracted from transfected cells and subjected to SDS-PAGE-Western blot assay. The USP18 antibody (Cell Signaling Technology, #4813) specifically binds to the USP18 protein, with β-actin used as an internal control. Results are shown below. Figure 12 As shown, the expression of USP18 protein in the NC-siRNA control group was significantly lower than that in the USP18-siRNA control group (P<0.0001), indicating that USP18-siRNA reduced intracellular USP18 levels.

[0104] Example 12 The NC-siRNA control group cells and USP18-siRNA control group cells in Example 11 were treated according to the hypoxia-reoxygenation modeling treatment method in Example 10, and NC-siRNA control group modeling cells and USP18-siRNA control group modeling cells were obtained respectively.

[0105] Caspase-3 enzyme activity was measured in NC-siRNA control group cells and USP18-siRNA control group cells, as well as in NC-siRNA control group model cells and USP18-siRNA control group model cells. The specific experimental steps are as follows: (1) Sample preparation: Collect myocardial tissue or cell samples and wash them with an appropriate buffer (such as PBS).

[0106] Place the sample on ice, add lysis buffer (usually containing protease inhibitors and phosphatase inhibitors), and homogenize or sonicate.

[0107] Centrifuge the sample to remove cell debris and collect the supernatant for testing.

[0108] (2) Detection method: The detection was performed using the fluorescent substrate Z-DEVD-AMC (a specific caspase-3 substrate).

[0109] When the sample is incubated with the Z-DEVD-AMC substrate at 37°C, caspase-3 specifically cleaves the substrate, releasing the fluorophore AMC.

[0110] Fluorescence intensity was measured using a fluorescence spectrophotometer or a fluorescence microplate reader at an excitation wavelength of 380 nm and an emission wavelength of 460 nm.

[0111] The activity of caspase-3 is calculated based on changes in fluorescence intensity, usually expressed as the rate of change in fluorescence intensity (e.g., change in fluorescence intensity per minute).

[0112] (3) Data processing: Changes in caspase-3 activity were assessed by comparing the changes in fluorescence intensity among different treatment groups.

[0113] like Figure 13 As shown, the Caspase-3 enzyme activity in the NC-siRNA control group was significantly lower than that in the USP18-siRNA control group (P < 0.0001). The Caspase-3 enzyme activity in the NC-siRNA control group was also significantly lower than that in the USP18-siRNA control group (P < 0.0001). Compared with the NC-siRNA control group, the activity of the USP18-siRNA control group was significantly lower, indicating reduced cell damage. Figure 13In the diagram, cells highlighted in blue by Ctrl represent NC-siRNA control group cells, cells highlighted in red by Ctrl represent USP18-siRNA control group cells, cells highlighted in blue by H / R represent NC-siRNA control group model cells, and cells highlighted in red by H / R represent USP18-siRNA control group model cells.

[0114] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Application of the USP18 gene as a drug target in screening drugs for the prevention, mitigation and / or treatment of myocardial ischemia-reperfusion injury.

2. The application as described in claim 1, characterized in that: The drug includes a USP18 gene expression inhibitor, which includes nucleic acid and / or adenovirus.

3. The application as described in claim 2, characterized in that: The nucleic acid includes siRNA, the sequence of which is shown in SEQ ID NO.6; And / or, the adenovirus includes AD-shRNA or adeno-associated virus AAV-shRNA.

4. Application of USP18 gene expression inhibitors in the preparation of drugs for the prevention, relief and / or treatment of myocardial ischemia-reperfusion injury.

5. The application as described in claim 4, characterized in that: The USP18 gene expression inhibitors include nucleic acids and / or adenoviruses.

6. The application as described in claim 5, characterized in that: The nucleic acid includes siRNA, the sequence of which is shown in SEQ ID NO.6; And / or, the adenovirus includes AD-shRNA or adeno-associated virus AAV-shRNA.

7. Application of PTEN-L neutralizing antibody in the preparation of drugs for the prevention, relief and / or treatment of myocardial ischemia-reperfusion injury.

8. A drug for preventing, alleviating, and / or treating myocardial ischemia-reperfusion injury, characterized in that: The drug includes one or more of the USP18 gene expression inhibitor and PTEN-L neutralizing antibody.

9. The drug as described in claim 8, characterized in that: The USP18 gene expression inhibitors include nucleic acids and / or adenoviruses.

10. The medicament as described in claim 9, characterized in that: The nucleic acid includes siRNA, the sequence of which is shown in SEQ ID NO.6; And / or, the adenovirus includes AD-shRNA or adeno-associated virus AAV-shRNA.

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