Application of TIF1beta in preparation of medicine for treating myocardial ischemia-reperfusion injury

Abstract

The invention provides an application of TIF1beta in preparation of a medicine for treating myocardial ischemia reperfusion injury. Silencing of the TIF1beta reduces injury of myocardial cells after myocardial ischemia reperfusion injury by reducing TRAF6 protein expression. The TIF1beta provided by the invention promotes the stability of the TRAF6 protein by mediating SUMOylation modification of the TRAF6 protein so as to enhance the expression of the TRAF6, thereby aggravating myocardial ischemia reperfusion injury by inhibiting the activity of myocardial cells and increasing the levels of apoptosis and inflammatory factors.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to an application of TIF1β in preparing a medicine for treating myocardial ischemia-reperfusion injury. Background Art

[0002] As we all know, acute myocardial infarction (AMI) is a common cardiovascular disease in the middle-aged and elderly population, and its high mortality rate claims the lives of millions of patients every year. At present, thrombolytic therapy and percutaneous coronary intervention to restore coronary blood flow as soon as possible are the main treatment options for AMI, known as myocardial reperfusion therapy, which reduces the scope of myocardial infarction to a certain extent and significantly improves the clinical prognosis of patients. It is worth noting that there is a lot of evidence that reperfusion sometimes leads to myocardial tissue damage and decreased cardiac function, manifested as acute heart failure, arrhythmia or sudden death. Myocardial ischemia-reperfusion injury (MIRI) refers to myocardial cell damage caused by high perfusion through blocked coronary arteries in a short period of time. Current studies have shown that the pathophysiological process of MIRI is extremely complex, and many key molecules are involved in the MIRI process. Therefore, starting from key molecules, exploring the regulatory mechanism behind MIRI and revealing its role in MIRI are of certain significance for preventing the occurrence of MIRI and developing molecular-targeted drugs to treat MIRI.

[0003] Tumor necrosis factor receptor-associated factor 6 (TRAF6), as a member of the TRAF family, acts as a multifunctional intracellular adaptor and E3 ubiquitin ligase. The role of TRAF6 in various diseases including the cardiovascular system has been more or less revealed, affecting disease progression by mediating a variety of biological processes such as inflammatory response, apoptosis and oxidative stress. TRAF6 plays an extremely important role in MIRI. However, whether the high expression of TRAF6 in the MIRI model is regulated by other molecules is worth exploring from other aspects. Summary of the invention

[0004] In view of this, the present invention aims to overcome the defects in the prior art and proposes the use of TIF1β in the preparation of a drug for treating myocardial ischemia-reperfusion injury.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] The present invention provides an application of TIF1β (E3 SUMO ligase) in preparing a drug for treating myocardial ischemia-reperfusion injury. Silencing of TIF1β reduces the damage of myocardial cells after myocardial ischemia-reperfusion injury by reducing the expression of TRAF6 protein.

[0007] Furthermore, the silencing of TIF1β increased the survival rate of cardiomyocytes by reducing the expression of TRAF6 protein and promoted the decrease of the levels of inflammatory factors in cardiomyocytes.

[0008] Furthermore, the silencing of TIF1β reduced the expression and stability of TRAF6 protein by regulating SUMOylation.

[0009] Furthermore, the silencing of TIF1β enhanced the cell viability of AC16 cells induced by hypoxia / reoxygenation by reducing the expression of TRAF6 protein and reduced cell apoptosis and inflammatory response.

[0010] Furthermore, overexpression of the TRAF6 protein reduced the cell viability of AC16 cells induced by hypoxia / reoxygenation and enhanced cell apoptosis and inflammatory response.

[0011] The present invention also provides an application of TIF1β in preparing a detection reagent for myocardial ischemia-reperfusion injury, wherein the silencing of TIF1β reduces the expression and stability of TRAF6 protein by regulating SUMOylation.

[0012] The present invention also provides an application of TIF1β in preparing a myocardial ischemia-reperfusion injury auxiliary diagnosis kit.

[0013] The present invention also provides a primer set for TIF1β, which comprises primers for TIF1β.

[0014] The present invention also provides a myocardial ischemia-reperfusion injury auxiliary diagnosis kit, which comprises the TIF1β primer set.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] The TIF1β described in the present invention promotes the stability of TRAF6 protein to enhance the expression of TRAF6 by mediating the SUMOylation modification of TRAF6 protein, thereby aggravating myocardial ischemia-reperfusion injury by inhibiting the vitality of cardiomyocytes and increasing the levels of apoptosis and inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The MI / R operation described in the embodiment of the present invention caused myocardial tissue damage and inflammation in mice: the mice were subjected to left anterior descending artery (LAD) occlusion for 30 minutes and then reperfused for 24 hours to establish a myocardial ischemia-reperfusion injury (MIRI) model; wherein, Figure A used HE staining to evaluate the morphology of myocardial tissue, Figure B used TTC staining to measure the size of myocardial infarction, Figure C detected the expression of Bax and Bcl-2 in heart tissue by Western blotting, and Figure DE used ELISA and RT-qPCR to study the levels of TNF-α and IL-6 in heart tissue; ***P<0.001;

[0018] Figure 2 The MI / R operation described in the embodiment of the present invention increased the expression of TRAF6 and SUMO1 in mice, and reduced the expression of SUMO2 and SUMO3: the mice were subjected to left anterior descending artery (LAD) occlusion for 30 minutes and then reperfused for 24 hours to establish a myocardial ischemia-reperfusion injury (MIRI) model; wherein, Figure A detected the expression of TRAF6 in heart tissue by RT-qPCR and Western blot, and Figure B detected the expression of SUMO1, SUMO2 and SUMO3 in heart tissue by Western blot; ***P<0.001;

[0019] Figure 3 H / R induction as described in the examples of the present invention promotes inflammatory response, apoptosis and TRAF6 expression in AC16 cells: AC16 cells were induced by hypoxia / reoxygenation (H / R). (A) ELISA was used to determine the levels of TNF-α and IL-6 in the cell supernatant; wherein, Figure A used ELISA to determine the levels of TNF-α and IL-6 in the cell supernatant, Figure B used RT-qPCR to determine the levels of intracellular TNF-α and IL-6, Figure C detected the expression of Bax and Bcl-2 in cardiac tissue by Western blot, and Figure D detected the expression of TRAF6 by RT-qPCR and Western blot; ***P<0.001;

[0020] Figure 4 The expression of SUMO1 described in the embodiment of the present invention is enhanced in AC16 cells induced by H / R and interacts with TRAF6 protein: AC16 cells were induced by hypoxia / reoxygenation (H / R); wherein, Figure A detected the expression of SUMO1, SUMO2 and SUMO3 by Western blotting, and Figure B verified the interaction between SUMO1 and TRAF6 in AC16 cells induced by H / R by co-immunoprecipitation (Co-IP); ***P<0.001;

[0021] Figure 5TIF1β as described in the embodiment of the present invention enhances the expression of TRAF6 by mediating the SUMOylation of TRAF6: wherein, Figure A shows an image of multiple SUMO modification sites of TRAF6 protein, Figure B predicts TIF1β-mediated SUMOylation of TRAF6 through the GPS-SUMO prediction website (https: / / sumo.biocuckoo.cn / ), Figure C verifies the interaction between TIF1β and TRAF6 in AC16 cells using Co-IP, Figure D detects the transfection efficiency by RT-qPCR, Figure E detects the expression of TIF1β in sh-TIF1β-transfected AC16 cells by western blot, and uses western blot to evaluate the stability of TRAF6 protein in sh-TIF1β-transfected AC16 cells after CHX treatment, and Figure F measures the level of TRAF6 in AC16 cells transfected with sh-TIF1β or pc-TIF1β by RT-qPCR and western blot; ***P<0.001;

[0022] Figure 6 TIF1β promotes myocardial cell injury by increasing TRAF6 expression as described in the examples of the present invention: AC16 cells were transfected with sh-TIF1β or co-transfected with pc-TRAF6, and then induced by hypoxia / reoxygenation (H / R); wherein, Figure A detected the transfection efficiency by RT-qPCR, Figure B detected the cell viability by CCK-8 kit, Figure C determined cell apoptosis by flow cytometry, and Figure D detected the TNF-α and IL-6 levels in the cell supernatant by ELISA. ***P<0.001. DETAILED DESCRIPTION

[0023] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0024] The present invention will be described in detail below with reference to the embodiments.

[0025] Example 1 Method

[0026] 1. Establishment and treatment of MIRI mice

[0027] Male C57BL / 6 mice (8-10 weeks old) were purchased from Hubei Experimental Animal Research Center. The animal experiments were approved by the Experimental Animal Ethics Committee of Tianjin Fifth Central Hospital. To detect TRAP6 expression in MIRI mice, the mice were divided into sham operation group and MIRI group. There were 5 mice in each group. For MIRI in mice, the mice were anesthetized with sodium pentobarbital. Subsequently, ischemia was induced by occluding the left anterior descending (LAD) artery for 30 minutes. Afterwards, blood flow to the LAD was restored. In the sham operation group, the mice underwent the same procedure but without ligation. After 24 hours of reperfusion, the mice were euthanized and the heart tissues were collected for subsequent experiments.

[0028] 2. Hematoxylin and eosin (HE) staining

[0029] The removed myocardial tissue was fixed in 4% paraformaldehyde, and then the tissue was embedded in paraffin blocks and cut into 5-μm-thick sections. Staining was performed with hematoxylin for 2 minutes and eosin for 30 seconds. Then, the sections were dehydrated using different gradient concentrations of ethanol. Images were observed using an Olympus microscope.

[0030] 3. Evans blue-2,3,5-triphenyltetrazolium chloride (TTC) staining

[0031] After 24 hours of perfusion, the mice were anesthetized and the left anterior descending (LAD) artery was reoccluded at the previously ligated position. Then, 1 ml of 1% Evans blue (Sigma-Aldrich, USA) was injected into the left ventricular cavity. The mouse heart was then quickly removed and washed. The heart was cut transversely into 1 mm thick slices. The slices were incubated with TTC solution (Solarbio, China) for 15 minutes. The stained slice images of each group were captured, and the infarct area of ​​each slice was measured using Image J software.

[0032] 4. Cell culture and treatment

[0033] Human AC16 cardiomyocytes were obtained from ATCC, USA. AC16 cells were cultured in DMEM (Thermo, USA) supplemented with 10% FBS (Thermo). AC16 cells were cultured in a humidified atmosphere containing 5% CO2 at a temperature of 37 degrees Celsius.

[0034] To establish a MIRI model in vitro, hypoxia / reoxygenation (H / R) conditions were used to treat AC16 cells. AC16 cells were placed in a hypoxic incubator (maintaining 5% CO2, 1% O2, and 94% N2) and exposed to hypoxia for 6 hours. After hypoxia treatment, the cells were transferred to a normoxic incubator (containing 95% air and 5% CO2) and reoxygenated at 37°C for 24 hours.

[0035] 5. Cell transfection

[0036] Short hairpin RNA targeting TIF1β (sh-TIF1β), overexpression plasmids including p-TIF1β and p-TRAF6, and control groups including sh-NC and p-NC were obtained from GenePharma, China. TM The plasmids were transfected into AC16 cells using Invitrogen 3000 (from Invitrogen, USA) for 48 hours.

[0037] 5. Enzyme-linked immunosorbent assay (ELISA)

[0038] TNF-α and IL-6 ELISA kits from FineTest (China) were used to determine the levels of inflammatory cytokines in mouse heart tissues and AC16 cell supernatants according to the manufacturer’s instructions.

[0039] 6. RT-qPCR

[0040] Total RNA was extracted from mouse heart tissue and AC16 cells using Trizol (Takara, Japan). Subsequently, total RNA was reverse transcribed into cDNA using Takara's RNA reverse transcription kit. Quantitative real-time PCR was performed on an ABI Prism 7500RT PCR system (Applied Biosystems, USA) using Takara's SYBR Premix Ex Taq. The relative expression of genes including TNF-α, IL-6, and TRAF6 was normalized to GAPDH by applying the 2-ΔΔCt formula.

[0041] 7. Protein immunoblotting

[0042] Mouse heart tissue and differently transfected AC16 cells were lysed using RIPA buffer (Biyuntian, China). The concentration of the extracted protein was determined by the BCA method. Subsequently, equal amounts of protein were loaded into the gel wells and then separated by electrophoresis. The separated proteins were transferred to a PVDF membrane. The PVDF membrane was incubated with skim milk (5%) for 1 hour. Afterwards, the PVDF membrane was incubated overnight at 4°C with primary antibodies against Bax (MA5-14003, 1:100), Bcl-2 (PA5-27094, 1:2000), TRAF6 (38-0900, 1:100), SUMO1 (MA5-14877, 1:1000), SUMO2 (PA5-110446, 1:1000), SUMO3 (700186, 1:1000), TIF1β (PA5-27648, 1:5000), β-actin (MA1-140, 1:10000) and GAPDH (PA1-987, 1:1000), all purchased from Thermo. The PVDF membrane was washed three times with TBST solution on the next day, and then the PVDF membrane was incubated with secondary antibodies for 2 hours. Finally, ECL chemiluminescence reagent (Thermo) was used to visualize protein bands, and grayscale values ​​were evaluated using Image J.

[0043] 8. Co-immunoprecipitation (Co-IP)

[0044] First, AC16 cells were lysed using cell lysate (Beyotime) to extract total protein. Subsequently, supernatant was obtained by centrifugation. Cell lysate was incubated overnight at 4°C with anti-SUMO1 (MA5-14877, Thermo), anti-TRAF6 (38-0900, Thermo) and anti-IgG (ab172730, Abcam). Afterwards, protein A / G agarose beads (SantaCruz, USA) were incubated for 4 hours at 4°C. After washing the samples five times, immunoprecipitation complexes were analyzed using Western blotting.

[0045] 9. Cycloheximide (CHX) determination method

[0046] To investigate the effect of TIF1β on TRAF6 protein stability, AC16 cells were transfected with sh-NC or sh-TIF1β and then treated with 100 μg / ml CHX (Sigma-Aldrich) for 0 h, 4 h, and 8 h. Afterwards, the TRAF6 protein levels at different time points during CHX treatment were quantified using Western blot analysis.

[0047] 10. Cell counting kit-8 (CCK-8) assay

[0048] AC16 cells were seeded into 96-well plates at a density of 1 × 10 4 Cells were added and then cultured overnight. Afterwards, 10 μl of CCK-8 solution (Biyuntian) was added to each well and incubated at 37°C for 2 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0049] 11. Flow cytometry

[0050] The apoptosis rate of AC16 cells was measured by flow cytometry. AC16 cells were cultured and collected. After washing with PBS, the cells were resuspended in 100 μL of 1× binding buffer and then stained with 10 μg / ml Annexin V / 5 μl propidium iodide (PI) double staining solution (Biyuntian) at 37°C for 15 minutes. Finally, the apoptosis rate was analyzed using a flow cytometer (Thermo Fisher).

[0051] 12. Statistical analysis

[0052] Data were analyzed using GraphPad Prism 9.0. Data are presented as mean ± standard deviation (SD). Unpaired Student's t test was used to compare two groups. For comparisons involving three or more groups, one-way analysis of variance (ANOVA) was applied, followed by Tukey's post hoc multiple comparison test. The threshold for statistical significance was set at p < 0.05.

[0053] Example 2 Results Analysis

[0054] 1. MI / R operation causes myocardial tissue damage and inflammation in mice

[0055] First, we established a myocardial ischemia / reperfusion (MI / R) model in mice by 30 minutes of left anterior descending coronary artery (LAD) occlusion followed by 24 hours of reperfusion. To verify the success of the mouse MI / R model, we performed several experiments. Figure 1 As shown in A, HE staining showed that the heart tissue of MI / R mice had inflammatory infiltration, structural disorder, and interstitial edema. In addition, we observed that MI / R surgery led to an increase in the size of myocardial infarction in mice, which was confirmed by TTC staining ( Figure 1 B). Subsequently, in cardiac tissue, we found that I / R surgery enhanced Bax expression and decreased Bcl-2 expression ( Figure 1 C), which indicates that I / R surgery induced cardiomyocyte apoptosis. In addition, by ELISA and RT-qPCR, we found that the levels of inflammatory factors such as TNF-α and IL-6 increased after MI / R surgery ( Figure 1 DE). In general, the MIRI mouse model was successfully established.

[0056] 2. MI / R operation increased the expression of TRAF6 and SUMO1 in mice, while reducing the expression of SUMO2 and SUMO3

[0057] As mentioned above, TRAF6 is associated with MIRI21. Here, TRAF6 expression was significantly increased in cardiac tissues of mice that underwent MI / R surgery compared with mice that did not undergo MI / R surgery ( Figure 2 A). At the same time, we found that the expression of SUMO1 was higher, while the expression of SUMO2 and SUMO3 was lower, in cardiac tissues after MI / R surgery compared with cardiac tissues without MI / R surgery ( Figure 2 B). Taken together, TRAF6 may play an important role in MIRI, and the increased expression of TRAF6 in MIRI mice may be related to SUMOylation.

[0058] 3. H / R induction promoted inflammatory response, apoptosis and TRAF6 expression in AC16 cells

[0059] In order to verify the abnormal high expression of TRAF6 in the MIRI cell model, AC16 cells were used as experimental subjects and induced by hypoxia / reoxygenation (H / R). Figure 3 As shown in A, ELISA assay showed abnormally increased levels of TNF-α and IL-6 in the supernatant of H / R-induced AC16 cells. Similarly, RT-qPCR revealed the same upward trend of TNF-α and IL-6 levels in H / R-induced AC16 cells as the ELISA results ( Figure 3 B). In addition, H / R induction increased the expression of Bax and attenuated the expression of Bcl-2 in AC16 cells ( Figure 3 C). More importantly, similar to the results in MIRI mice, TRAF6 expression was significantly increased in H / R-induced AC16 cells ( Figure 3 D). In summary, TRAF6 is closely related to MIRI.

[0060] There is an interaction between SUMO1 protein and TRAF6 protein, which is reflected by the successful pull-down of TRAF6 protein by SUMO1 protein antibody ( Figure 4 B). Moreover, H / R induction promoted the interaction between these two proteins ( Figure 4 B) Therefore, we considered that TRAF6 might contribute to H / R-induced SUMOylation in AC16 cells.

[0061] 4. TIF1β enhances the expression of TRAF6 by mediating SUMOylation of TRAF6

[0062] Through bioinformatics analysis, we observed multiple SUMO modification sites in TRAF6 protein ( Figure 5 A). We predicted that SUMO E3 ligase TIF1β may mediate SUMOylation of TRAF6 through the GPS-SUMO prediction website (https: / / sumo.biocuckoo.cn / ) Figure 5 B). In addition, the interaction between TIF1β and TRAF6 was verified by Co-IP experiments, in which TRAF6 antibody pulled down TIF1β protein ( Figure 5 C). Then, the stability of TRAF6 protein mediated by TIF1β in AC16 cells was examined after CHX treatment. Of note, AC16 cells were transfected with sh-NC or sh-TRAF6 and then treated with CHX ( Figure 5 D). First, we observed a decrease in TIF1β expression in AC16 cells transfected with sh-TIF1β. Then, we found that TRAF6 knockdown attenuated the stability of TRAF6 protein in AC16 cells ( Figure 5 E). In addition, TRAF6 knockdown reduced TRAF6 levels, whereas TRAF6 overexpression enhanced TRAF6 levels ( Figure 5 F). The above results indicate that TIF1β increases TRAF6 protein expression by mediating SUMOylation of TRAF6.

[0063] 5. TIF1β promotes cardiomyocyte injury by increasing TRAF6 expression

[0064] To investigate how the TIF1β / TRAF6 axis affects cardiomyocyte injury, AC16 cells were transfected with sh-TIF1β or co-transfected with pc-TRAF6 ( Figure 6 A). Then, the transfected AC16 cells were subjected to hypoxia / reoxygenation (H / R) induction. Here, CCK-8 assay showed that H / R induction suppressed cell viability, which was alleviated by TIF1β knockdown. However, TRAF6 overexpression abolished the effect of TIF1β knockdown on the H / R-induced increase in cell viability of AC16 cells ( Figure 6 B). In contrast, we observed a significant increase in apoptosis in H / R-induced AC16 cells compared with normal AC16 cells. As expected, TIF1β knockdown inhibited H / R-induced apoptosis in AC16 cells, while TRAF6 overexpression counteracted the inhibitory effect of TIF1β knockdown on apoptosis ( Figure 6C). In addition, TIF1β knockdown reduced the upregulation of TNF-α and IL-6 levels in the supernatant of AC16 cells induced by H / R. However, the decrease in TNF-α and IL-6 levels in AC16 cells induced by H / R due to TIF1β knockdown was reversed by TRAF6 overexpression ( Figure 6 D). In summary, TIF1β knockdown inhibited TRAF6 expression, enhanced cell viability, and reduced apoptosis as well as TNF-α and IL-6 levels, thereby alleviating cardiomyocyte damage after myocardial ischemia-reperfusion injury (MIRI).

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of TIF1β in the preparation of a drug for treating myocardial ischemia-reperfusion injury, characterized in that: Silencing of TIF1β alleviated cardiomyocyte damage after myocardial ischemia-reperfusion injury by reducing the expression of TRAF6 protein.

2. The use according to claim 1, characterized in that: The silencing of TIF1β increases the survival rate of cardiomyocytes by reducing the expression of TRAF6 protein and promotes the decrease of the levels of inflammatory factors in cardiomyocytes.

3. The use according to claim 1, characterized in that: The silencing of TIF1β reduced the expression and stability of TRAF6 protein by regulating SUMOylation.

4. The use according to claim 1, characterized in that: The silencing of TIF1β enhanced the cell viability of AC16 cells induced by hypoxia / reoxygenation by reducing the expression of TRAF6 protein and reduced cell apoptosis and inflammatory response.

5. The use according to claim 4, characterized in that: Overexpression of the TRAF6 protein reduced the cell viability of AC16 cells induced by hypoxia / reoxygenation and enhanced cell apoptosis and inflammatory response.

6. Application of TIF1β in the preparation of a detection reagent for myocardial ischemia-reperfusion injury, characterized in that: The silencing of TIF1β reduced the expression and stability of TRAF6 protein by regulating SUMOylation.

7. Application of TIF1β in the preparation of auxiliary diagnostic kit for myocardial ischemia-reperfusion injury.

8. A primer set for TIF1β, characterized in that: The primer set includes a primer for TIF1β.

9. A myocardial ischemia-reperfusion injury auxiliary diagnosis kit, characterized in that: The kit comprises the TIF1β primer set as claimed in claim 8.

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