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

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of an INSR inhibitor to preparation of a medicine for treating myocardial ischemia-reperfusion injury. The invention provides INSR, which is a therapeutic target for myocardial ischemia reperfusion injury (MIRI), and also provides application of NVP-ADW742 or dapagliflozin as an INSR inhibitor in preparation of a therapeutic drug for treating myocardial ischemia reperfusion injury, and the NVP-ADW742 and dapagliflozin play a role in protecting myocardial cells by inhibiting expression of INSR and restoring autophagic circulation of ischemia reperfusion injury. Therefore, the invention provides a novel therapeutic target and a novel therapeutic method for MIRI, and has great significance for researching and treating myocardial ischemia reperfusion injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of an INSR inhibitor in the preparation of a drug for treating myocardial ischemia-reperfusion injury. Background Art

[0002] Myocardial ischemic disease caused by coronary artery disease is one of the leading causes of morbidity and mortality worldwide. Although timely restoration of myocardial tissue blood perfusion is the most effective treatment for myocardial infarction, it can also lead to additional cardiomyocyte death and aggravate cardiac function impairment, ultimately leading to myocardial ischemia-reperfusion injury (MIRI). The mechanism of MIRI is complex, and currently discovered mechanisms include inflammation, programmed cell death, mitochondrial dysfunction, calcium overload, and endothelial cell dysfunction. The clinical treatment effects of intervention measures based on these mechanisms are not ideal, so further in-depth research on the potential mechanisms of MIRI and the search for new therapeutic targets are needed.

[0003] Insulin is an essential hormone that controls the availability of glucose required for nearly all cellular processes. Insulin is widely involved in the regulation of cardiac function, including cardiac metabolism, cardiomyocyte survival, and cardiac growth. The insulin receptor (INSR) is highly abundant in the heart. Insulin promotes glucose uptake into cardiomyocytes by activating the insulin receptor-mediated phosphatidylinositol 3-kinase (PI3K)-Akt pathway. This pathway mobilizes glucose transporter 4 (GLUT4) vesicles to the cell surface for glucose uptake.

[0004] Previous studies have shown that insulin receptor signaling is closely related to atherosclerosis and heart failure. mTOR is a serine / threonine kinase that belongs to the PI3K-related kinase (PIKK) family. It can interact with several proteins to form two different complexes, called mTOR complex 1 (mTORC1) and 2 (mTORC2). mTORC1 inactivates the formed autophagy regulatory complex through phosphorylation, thereby inhibiting autophagy. Current studies have shown that InsR / IGF1R promotes macrophage polarization to M1-like through mTORC1, thereby manifesting as overall pro-inflammatory. At the same time, autophagy promotes oxidative phosphorylation (OXPHOS) through lipid catabolism. After mTORC1 is activated, lipid catabolism is inhibited, exacerbating the occurrence and development of atherosclerosis. Myocardial ischemia-reperfusion injury is an important process of coronary atherosclerosis and an important cause of heart failure. Research on insulin receptor and myocardial ischemia-reperfusion injury is still lacking. Summary of the invention

[0005] To solve the above problems, one of the purposes of the present invention is to provide a therapeutic target for myocardial ischemia-reperfusion injury (MIRI), which is INSR. The present invention has experimentally demonstrated that the expression of INSR protein increases after myocardial cell ischemia-reperfusion, and inhibiting INSR expression has an improving effect on MIRI.

[0006] The second object of the present invention is to provide an application of an INSR inhibitor in the preparation of a therapeutic drug for treating myocardial ischemia-reperfusion injury.

[0007] Furthermore, the INSR inhibitor is NVP-ADW742 or dapagliflozin.

[0008] Furthermore, NVP-ADW742 or dapagliflozin inhibits the expression of INSR, thereby restoring the smooth flow of autophagy in ischemia-reperfusion injury and protecting cardiomyocytes.

[0009] Furthermore, the drug for treating myocardial ischemia-reperfusion injury comprises an active ingredient, wherein the active ingredient is an INSR inhibitor, and the INSR inhibitor is NVP-ADW742 or dapagliflozin.

[0010] Furthermore, the myocardial ischemia-reperfusion injury therapeutic drug also includes a pharmaceutically acceptable carrier, and "pharmaceutically acceptable" means that when the carrier is properly administered to an animal or a human, they will not produce adverse, allergic or other adverse reactions. The pharmaceutically acceptable carrier includes, but is not limited to, one or more of a diluent, a filler, a surfactant, an absorption promoter, a disintegrant, a wetting agent, and a dispersant.

[0011] Furthermore, the dosage form of the drug for treating myocardial ischemia-reperfusion injury includes but is not limited to any one of injection, tablet, powder, granule, pill, capsule, and oral agent.

[0012] The present invention has the following beneficial effects:

[0013] 1. The present invention proves at the cellular level that the expression of INSR protein increases after myocardial cell ischemia-reperfusion.

[0014] 2. The present invention confirms that NVP-ADW742 has a protective effect on myocardial cells by inhibiting the expression of INSR and restoring the smooth autophagic flow of ischemia-reperfusion injury. It also confirms that dapagliflozin also inhibits the expression of INSR and has a similar protective effect on myocardial ischemia-reperfusion injury.

[0015] 3. The present invention confirms that overexpression of INSR aggravates myocardial ischemia-reperfusion injury, which in turn proves the significance of INSR in myocardial ischemia-reperfusion injury.

[0016] The above results indicate that INSR is a potential therapeutic target for the treatment of MIRI, which provides a research basis for the application of INSR inhibitors in the preparation of therapeutic drugs for myocardial ischemia-reperfusion injury, and is of great significance for the research and treatment of myocardial ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : In Example 1, Western Blot was used to detect the expression level of INSR in H / N cardiomyocytes.

[0018] Figure 2 : Viability of H / N cardiomyocytes treated with NVP-ADW742 and DMSO in Example 2. In the figure, **: H / N-DMSO vs. N-NVP-ADW742; ##: H / N-NVP-ADW742 vs. H / N-DMSO, N=6 / group.

[0019] Figure 3 : Staining results of apoptosis and necrosis of H / N cardiomyocytes treated with NVP-ADW742 and DMSO in Example 2, Scale bar 200 μm, N=4 / group.

[0020] Figure 4 : In Example 2, Western Blot was used to detect the expression of autophagy-related proteins in H / N cardiomyocytes treated with INSR inhibitor NVP-ADW742 and DMSO.

[0021] Figure 5 : Fluorescence staining of autophagic flux in H / N cardiomyocytes treated with INSR inhibitor NVP-ADW742 and DMSO in Example 2, Scale bar 200 μm, N=4 / group.

[0022] Figure 6 : In Example 3, Western Blot was used to detect the INSR overexpression effect after myocardial cells were transfected with INSR mode RNA and overexpression adenovirus.

[0023] Figure 7 : H / N cardiomyocyte viability of INSR overexpression and control cardiomyocytes treated with dapagliflozin and DMSO in Example 3. Left: INSR modeRNA overexpression, **: LNP-DAPA VS LNP-DMSO, ##: INSR-DAPA VS LNP-DAPA, N=6 / group; Right: Ad-INSR overexpression, PA, N=6 / group. *: CON-DAPA VS CON-DMSO, ##: INSR-DAPA VS CON-DA.

[0024] Figure 8: Staining of apoptosis and necrosis of H / N cardiomyocytes treated with dapagliflozin and DMSO in INSR overexpressed and control cardiomyocytes in Example 3. Top: INSR modeRNA overexpression; Bottom: Ad-INSR overexpression. Scale bar 200 μm, N=4 / group.

[0025] Fig. 9 : Western Blot detection of the expression of autophagy-related proteins in H / N cardiomyocytes treated with dapagliflozin and DMSO in INSR overexpressed and control cardiomyocytes in Example 3. Top: INSR modeRNA overexpression; Bottom: Ad-INSR overexpression.

[0026] Fig.10 : Western Blot detection of autophagic flux fluorescence staining of INSR overexpressing and control cardiomyocytes in H / N cardiomyocytes treated with dapagliflozin and DMSO in Example 3, Scale bar 200 μm, N=4 / group. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0028] Example 1: Detection of INSR protein expression level after myocardial cell ischemia-reperfusion 1.1 Research subject: human myocardial cell line AC16.

[0029] 1.2 Cell culture: The cell culture conditions are DMEM medium + 10% FBS, 37°C, 5% CO2, saturated humidity. Cells in the growth state and with a cell density of 90% are taken for treatment.

[0030] 2. Experimental groups: (1) normoxia group (N+DMSO); (2) normoxia + dapagliflozin group (N+DAPA); (3) hypoxia-reoxygenation group (H / N+DMSO); (4) hypoxia-reoxygenation + dapagliflozin group (H / N+DAPA).

[0031] 3. Experimental methods:

[0032] (1) Establishment of the hypoxia-reoxygenation model of cardiomyocytes: AC16 cells were cultured to a density of about 90%, and the cells were placed in a hypoxic box (99% N2, <0.1% O2) for 1 hour, which is the hypoxia process (H). The cells were removed and the original culture medium was discarded, replaced with DMEM medium + DMSO (1:2000), and placed in a normal incubator (37°C, 5% CO2) for 1 hour, which is the reoxygenation process (R). The hypoxia-reoxygenation model simulates the state of cardiomyocytes after ischemia and reperfusion.

[0033] (2) Dapagliflozin treatment of cardiomyocytes: For cells under normoxic conditions and at a cell density of 90% or for cells at a cell density of 90% after 1 hour of hypoxia, the original culture medium was discarded, the cells were rinsed twice with PBS and then discarded. The culture medium was replaced with DMEM containing dapagliflozin (1:2000) and treated for 1 hour.

[0034] (3) Extraction of cell protein: discard the cell culture medium, wash once with PBS, add an appropriate amount of RIPA, scrape the cells with a cell scraper on ice, collect them into an Eppendorf tube, centrifuge at 16,000 g for 10 min at 4°C, discard the precipitate, transfer the supernatant to a new pre-cooled Eppendorf tube, determine the protein concentration by BCA method, add 1 / 4 volume of 5X protein loading, and store in the freezer.

[0035] 4. Experimental results: Figure 1 As shown in the figure, compared with the normoxia group, the expression of INSR in myocardial cells in the hypoxia-reoxygenation group was increased, while the addition of dapagliflozin inhibited the expression of INSR. mTOR, p62, and LC3B are key regulatory proteins in the autophagy process, and they play important roles in the initiation of autophagy, selective degradation, and autophagosome formation. Figure 1 It can be seen that compared with the normoxia group, the expression of p-mTOR and P62 in cardiomyocytes in the hypoxia-reoxygenation group was increased, and the LC3BII / I ratio was decreased. Compared with DMSO (i.e., the control group), dapagliflozin reduced the expression of p-mTOR and P62 and increased LC3BII / I, indicating that dapagliflozin promoted autophagy of cardiomyocytes after hypoxia-reoxygenation.

[0036] Example 2: Experimental study on the effect of NVP-ADW742 on myocardial ischemia-reperfusion injury

[0037] 1.1 Research subjects: Human cardiomyocyte cell line AC16.

[0038] 1.2 Cell culture: The cell culture conditions are DMEM medium + 10% FBS, 37°C, 5% CO2, saturated humidity, and cells in growth state and with a cell density of 90% are taken for treatment.

[0039] 2. Experimental groups: (1) normoxia group (N+DMSO); (2) normoxia+NVP-ADW742 group (N+NVP-ADW742); (3) hypoxia-reoxygenation group (H / N+DMSO); (4) hypoxia-reoxygenation+NVP-ADW742 group (H / N+NVP-ADW742).

[0040] 3. Experimental methods:

[0041] (1) Establishment of myocardial cell hypoxia-reoxygenation model: see Example 1.3(1).

[0042] (2) NVP-ADW742 treatment of cardiomyocytes: The cells were kept in normoxic conditions throughout the whole process, and the cells were kept at a cell density of 90% or the cells were kept at a cell density of 90% and then hypoxic for 1 hour. The original culture medium was discarded, and the cells were rinsed with PBS twice and then discarded. The culture medium was replaced with DMEM culture medium containing NVP-ADW742 (1:2000) and treated for 1 hour.

[0043] (3) Extraction of cell proteins: see Example 1.3(3).

[0044] (4) CellTiter-Lumi TM II luminescence method to detect cell viability: cells were seeded in a 96-well plate, and when the density reached 90%, they were allowed to stand at room temperature for 10 minutes. 100ul CellTiter-Lumi TM II luminescent detection reagent, shake for 2 minutes to promote cell lysis, incubate at room temperature for 10 minutes to stabilize the luminescent signal, and use a multi-function plate reader for chemiluminescent detection.

[0045] (5) Annexin V / PI double staining method to detect cardiomyocyte apoptosis and necrosis: a. Collect cells: remove the cells, discard the original culture medium, rinse the cells twice with PBS and then discard. Add 1 mL of trypsin and digest at 37°C for 1 min 30 s. When the cells are fully digested, add 2 mL of culture medium to stop digestion. Transfer the liquid in the culture flask into a centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant and keep the cell pellet; b. Wash the cells: add pre-cooled PBS to make a cell suspension, centrifuge at 1000 rpm for 5 min and discard the supernatant. Repeat the operation once; c. Resuspend the cells: add 1×Binding Buffer to make a cell suspension (concentration ≈1×10 6 / mL). Transfer 100μL of cell suspension to the flow tube; d. Annexin V-FITC labeling: add 5μL Annexin V-FITC under light-proof conditions and mix the cell suspension, incubate at room temperature for 15min; e. PI labeling: add 5μL PI staining 5min before loading; f. Preparation for loading: add 400μL 1× Binding Buffer. Perform flow cytometric analysis within 1h.

[0046] (6) mRFP-GFP-LC3 detection of autophagic flux: The target cells in good condition were inoculated into a 24-well plate, with the number of cells in each well being approximately 1.5×10 5, and cultured overnight in a 37°C, 5% CO2 incubator. Before infection, take out the virus from the refrigerator and slowly melt it on ice, remove the original culture medium of the cells, add 1 / 2 volume of fresh culture medium, and add the virus for infection. After 24 hours, remove the culture medium containing the virus, replace it with fresh complete culture medium, and continue to culture at 37°C for 48 hours. Turn on the confocal microscope, detect the fluorescence signal in the cells in the confocal dish and take pictures, detect the red fluorescence of mRFP protein at an excitation wavelength of 561nm and an emission wavelength of 570-620nm; detect the green fluorescence of GFP protein at an excitation wavelength of 488nm and an emission wavelength of 500-550nm. In the green and red merged image, the autophagosome is displayed as a yellow spot, and the autophagolysosome formed after the fusion of the autophagosome and the lysosome is displayed as a red spot; the autophagic flow can be observed by the ratio of yellow and red spots in the merge image.

[0047] 4. Experimental results: CellTiter-Lumi TM II luminescence assay to detect cell viability, such as Figure 2 As shown in Figure 2, compared with the normoxia group, myocardial cell viability decreased after hypoxia-reoxygenation, and NVP-ADW742 treatment reversed myocardial cell injury. Myocardial cell apoptosis and necrosis were detected by Annexin V / PI double staining. Figure 3 As shown in the figure, compared with H / N treated with DMSO, NVP-ADW742 reduced the necrosis and apoptosis of cardiomyocytes after hypoxia and reoxygenation. mTOR, p62, and LC3B are key regulatory proteins in the autophagy process, and they play important roles in different links such as the initiation of autophagy, selective degradation, and autophagosome formation. Figure 4 As shown in the figure, compared with the normoxia group, the expression of p-mTOR and P62 in the myocardial cells of the hypoxia-reoxygenation group increased, and the LC3BII / I ratio decreased. Compared with DMSO (i.e., the control group), NVP-ADW742 reduced the expression of p-mTOR and P62 and increased LC3BII / I, indicating that NVP-ADW742 promoted autophagy in myocardial cells after hypoxia-reoxygenation. The autophagy flow was detected by mRFP-GFP-LC3, as shown in the figure. Figure 5 As shown in the figure, hypoxia-reoxygenation increased the number of autophagosomes in cardiomyocytes, but reduced the number of autophagolysosomes, and the autophagic flow was blocked. NVP-ADW742 increased the formation of autophagolysosomes and restored the smooth autophagic flow after hypoxia-reoxygenation.

[0048] Example 3: Effect of dapagliflozin on myocardial ischemia-reperfusion injury Experiment 1.1 Study subject: human myocardial cell line AC16.

[0049] 1.2 Cell culture: The cell culture conditions were DMEM medium + 10% FBS, 37°C, 5% CO2, saturated humidity. After 48 hours, the cells treated as described below were placed in an oxygen-free chamber (99% N2, <0.1% O2) for 1 hour, which was the oxygen-free process (H). The cells were removed and the original culture medium was discarded, replaced with DMEM medium + DMSO / dapagliflozin (1:2000), and placed in a normal incubator (37°C, 5% CO2) for 1 hour, which was the reoxygenation process (R).

[0050] 2. Experimental groups: (1) control group (CON-DMSO); (2) INSR overexpression group (INSR-DMSO); (3) dapagliflozin group (CON-DAPA); (4) dapagliflozin + INSR overexpression group (INSR-DAPA).

[0051] 3. Experimental methods:

[0052] (1) Overexpression of INSR:

[0053] Adenovirus overexpression: Add the overexpression adenovirus (Ad-INSR) to the cell culture medium at a dilution ratio of 1:1000 (MOI:100), change the medium after 6 hours, and detect protein levels, cell viability, and autophagy after 48 hours. ModeRNA overexpression: Incubate modeRNA with transfection reagent for 10-15 minutes to form an mRNA-transfection complex. Gently add the complex to a cell culture dish with 50% cell density and mix well. Detect protein levels, cell viability, and autophagy after 48 hours.

[0054] (2) Establishment of the cardiomyocyte hypoxia-reoxygenation model: The cells treated as above were placed in a hypoxic chamber (99% N2, <0.1% O2) and cultured for 1 h, which was the hypoxia process (H). The cells were removed and the original culture medium was discarded, replaced with DMEM medium + DMSO (1:2000), and placed in a normal incubator (37°C, 5% CO2) and incubated for 1 h, which was the reoxygenation process (R).

[0055] (3) Dapagliflozin treatment of cardiomyocytes: After the cells treated above were subjected to 1 hour of hypoxia, the original culture medium was discarded, the cells were rinsed with PBS twice and then discarded. The culture medium was replaced with DMEM culture medium containing dapagliflozin (1:2000) and treated for 1 hour.

[0056] (4)WB, CellTiter-Lumi TM II luminescence method was used to detect cell viability, Annexin V / PI double staining method was used to detect cardiomyocyte apoptosis and necrosis, and mRFP-GFP-LC3 was used to detect autophagic flux: the experimental methods were as described above.

[0057] 4. Experimental results: Figure 6As shown, the expression level of INSR was detected by WB. Both modeRNA and overexpression adenovirus were used to overexpress INSR in cardiomyocytes. Figure 7 As shown in Figure 2, overexpression of INSR reduced cardiomyocyte viability, but dapagliflozin had a protective effect on cardiomyocytes overexpressing INSR modeRNA. Cardiomyocyte apoptosis and necrosis were detected by Annexin V / PI double staining. Figure 8 As shown in Figure 2, compared with ischemic reperfusion cardiomyocytes overexpressing INSR, dapagliflozin reduced necrosis and apoptosis of cardiomyocytes overexpressing INSR. mTOR, p62, and LC3B are key regulatory proteins in the autophagy process, and they play important roles in different links such as the initiation of autophagy, selective degradation, and autophagosome formation. Fig. 9 As shown in Figure 2, compared with the INSR overexpression group, dapagliflozin reduced the expression of p-mTOR and P62 and increased LC3BII / I, indicating that dapagliflozin reversed the effect of increased INSR on myocardial autophagy. Autophagic flow was detected by mRFP-GFP-LC3, as shown in Figure 2. Fig.10 As shown in the figure, overexpression of INSR increased the number of autophagosomes in cardiomyocytes, but reduced the number of autophagolysosomes, thus impeding the autophagic flux. Dapagliflozin increased the formation of autophagolysosomes and restored the smooth autophagic flux after overexpression of INSR.

[0058] This specific implementation is only an explanation of the present invention, not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. Application of INSR inhibitors in the preparation of therapeutic drugs for the treatment of myocardial ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that: The INSR inhibitor is NVP-ADW742 or dapagliflozin.

3. The use according to claim 2, characterized in that: The NVP-ADW742 or dapagliflozin inhibits the expression of INSR, restores the smooth flow of autophagy caused by ischemia-reperfusion injury, and protects myocardial cells.

4. The use according to claim 2, characterized in that: The myocardial ischemia-reperfusion injury therapeutic drug comprises an effective ingredient, and the effective ingredient is an INSR inhibitor.

5. The use according to claim 4, characterized in that: The drug for treating myocardial ischemia-reperfusion injury also includes a pharmaceutically acceptable carrier.

6. The use according to claim 4, characterized in that: The dosage form of the drug for treating myocardial ischemia-reperfusion injury is any one of injection, tablet, powder, granule, pill, capsule and oral preparation.

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