Use of phytochelatin 4 in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury

Abstract

The application discloses application of Phytochelatin 4 in preparation of a medicine for preventing and treating myocardial ischemia-reperfusion injury, and belongs to the technical field of biological medicine. After neonatal rat myocardial cells are treated by Phytochelatin 4 and then subjected to hypoxia-reoxygenation, a myocardial ischemia-reperfusion injury model is constructed in vitro, and it is found that the use of Phytochelatin 4 reduces the expression level of GRP78, reduces the number of apoptotic cells, reduces the calcium ion level, and relieves the endoplasmic reticulum stress degree, which indicates that Phytochelatin 4 plays a protective role on myocardial ischemia-reperfusion injury by regulating oxidative stress, calcium overload and endoplasmic reticulum stress, and this provides a new mechanism and strategy for prevention and treatment of myocardial ischemia-reperfusion injury (especially hypoxia-reoxygenation injury).

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of Phytochelatin 4 in the preparation of drugs for the prevention and treatment of myocardial ischemia-reperfusion injury. Background Technology

[0002] Acute myocardial infarction is a leading cause of death and high morbidity worldwide. Although thrombolysis and percutaneous coronary intervention can maximize the salvage of ischemic myocardium and reduce infarct size, reperfusion itself can induce additional damage, leading to cardiomyocyte death, microcirculatory disturbances, reperfusion arrhythmias, and further deterioration of cardiac function. Therefore, mitigating myocardial ischemia-reperfusion injury remains a key factor in improving patient prognosis. Previous studies have shown that multiple pathways, including oxidative stress, calcium overload, apoptosis, and endoplasmic reticulum stress, interact in this process.

[0003] In recent years, enhancing cellular antioxidant capacity, maintaining protein homeostasis, and alleviating endoplasmic reticulum stress have become important directions for the intervention of myocardial ischemia / reperfusion injury (MI / RI). Phytochelatin 4 is a heavy metal chelating peptide derived from glutathione, and existing literature and publicly available information mainly focus on its application in heavy metal stress, plant metal homeostasis, and analytical detection. Studies have found that in myocardial ischemia / reperfusion injury, cardiomyocytes exhibit significant endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) activation, manifested as upregulation of molecules such as glucose-regulated protein 78 (GRP78) and activated transcription factor 6 (ATF6), and unfolded protein aggregation. This process is closely related to cardiomyocyte apoptosis, inflammatory response, and adverse remodeling, suggesting that ERS is an important pathological link in ischemia / reperfusion injury. Among them, GRP78 is an important endoplasmic reticulum molecular chaperone protein that plays a core role in cellular stress response, protein folding, and quality control. Under physiological conditions, GRP78 assists in the correct folding of unfolded or misfolded proteins by binding to them and participates in the regulation of endoplasmic reticulum (ER) to maintain endoplasmic reticulum homeostasis. When cells face stress injuries such as hypoxia, GRP78 expression is upregulated, initiating the unfolded protein response (UPR) to help cells adapt to stress or induce apoptosis. However, there are no reports on the protective role of Phytochelatin 4 in myocardial ischemia-reperfusion injury, especially regarding the mechanism by which it exerts its cardioprotective effect through the regulation of oxidative stress, calcium overload, and ER stress, which lacks systematic research. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Phytochelatin 4 in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury, thereby solving the problems existing in the prior art. By constructing a simulated myocardial ischemia-reperfusion injury model in vitro using cardiomyocytes treated with Phytochelatin 4 after hypoxia-reoxygenation, the results showed that Phytochelatin 4 treatment reduced the expression level of GRP78, decreased the number of apoptotic cells, reduced calcium ion levels, and alleviated endoplasmic reticulum stress. This indicates that Phytochelatin 4 exerts a protective effect against myocardial ischemia-reperfusion injury by regulating oxidative stress, calcium overload, and endoplasmic reticulum stress. This provides a new mechanism and strategy for the prevention and treatment of myocardial ischemia-reperfusion injury (especially hypoxia-reoxygenation injury).

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of Phytochelatin 4 in the preparation of drugs for treating myocardial hypoxia-reoxygenation injury.

[0006] This invention also provides the use of Phytochelatin 4 in the preparation of drugs for preventing and treating myocardial ischemia-reperfusion injury.

[0007] Preferably, Phytochelatin 4 achieves its anti-myocardial hypoxia-reoxygenation injury or prevention of myocardial ischemia-reperfusion injury by inhibiting cardiomyocyte apoptosis.

[0008] Preferably, Phytochelatin 4 achieves its effect of resisting myocardial hypoxia-reoxygenation injury or preventing myocardial ischemia-reperfusion injury by alleviating myocardial cell calcium overload induced by hypoxia-reoxygenation.

[0009] Preferably, Phytochelatin 4 achieves its effect of resisting myocardial hypoxia-reoxygenation injury or preventing myocardial ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress in cardiomyocytes.

[0010] Preferably, Phytochelatin 4 inhibits endoplasmic reticulum stress by downregulating the expression of endoplasmic reticulum stress marker proteins GRP78, ATF6, p-IRE1α, and / or CHOP.

[0011] Preferably, Phytochelatin 4 achieves its anti-myocardial hypoxia-reoxygenation injury or prevention of myocardial ischemia-reperfusion injury by reducing GRP78 expression levels.

[0012] The present invention also provides a drug for preventing myocardial ischemia-reperfusion injury, the active ingredient of which includes Phytochelatin 4.

[0013] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0014] The present invention discloses the following technical effects: This invention discovers a novel use for Phytochelatin 4, revealing for the first time through experiments its protective effect against myocardial ischemia-reperfusion injury. Phytochelatin 4 can significantly alleviate myocardial ischemia-reperfusion injury, especially hypoxia-reoxygenation injury. Specifically, treatment with Phytochelatin 4 significantly reduces cardiomyocyte apoptosis, lowers calcium ion levels, and alleviates endoplasmic reticulum oxidative stress. Therefore, this invention elucidates a new mechanism for preventing and treating myocardial ischemia-reperfusion injury and demonstrates that Phytochelatin 4 can be used to prepare drugs for preventing and treating myocardial ischemia-reperfusion injury, especially hypoxia-reoxygenation injury. This has significant implications for subsequent theoretical research and clinical applications of Phytochelatin 4 in myocardial ischemia-reperfusion injury.

[0015] In summary, this invention provides a novel and effective small molecule intervention strategy for the prevention and treatment of myocardial ischemia-reperfusion injury, which has good scientific research value and clinical translation potential. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This diagram illustrates the binding mode of the MOUSE GRP78 protein to PC4. A: Cartoon image (left) and 3D image (right) of the MOUSE GRP78 protein binding to PC4; in the 3D image, the C backbone of the MOUSE GRP78 protein is shown in green, N atoms in blue, O atoms in bright red, H atoms in white, and PC4 is shown as a blue stick. B: Surface image (left) and 2D image (right) of the MOUSE GRP78 protein binding to PC4. Figure 2 These are the results of the CCK8 experiment; Figure 3 Effects of different concentrations of PC4 on oxidative stress response in the endoplasmic reticulum of primary cardiomyocytes; A: Western Blot results; B: Statistical graphs; Figure 4 The effect of different hypoxia-reoxygenation times on the endoplasmic reticulum oxidative stress response of primary cardiomyocytes; A: Western blotting results; B: Statistical graphs. Figure 5 Western blot analysis of changes in endoplasmic reticulum oxidative stress-related proteins in four groups of primary cardiomyocytes (A) and statistical data (B). Figure 6 Microscopic observation of calcium ion changes in four groups of primary cardiomyocytes (A) and statistical data (B). Figure 7 Microscopic observation of TUNEL fluorescence staining of four groups of primary cardiomyocytes (A) and statistical comparison of the percentage of TUNEL positive cell nuclei in the four groups (B). Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The following examples involve Phytochelatin 4 (PC4), whose molecular formula is C0. 34 H53 N9O 18 S4, with a molecular weight of 1004.09 Da and CAS number 99465-98-2, has the following chemical structure: The Phytochelatin 4 stock solution (MedChemExpress, China) purchased by this invention was used for experiments.

[0024] Example 1: Molecular docking Open DMFold, input the corresponding amino acid sequences of the two interacting proteins, IMPAD1 and GRP78, in FASTA format, run the docking test, and download the .pdb file of the interaction prediction model. Open the results file and the protein molecules, display the docking results based on the binding energy, and visualize the docking using Pymol.

[0025] The results showed that GRP78 interacts with IMPAD1.

[0026] Example 2: Cell Model Construction 1. Construct a primary model of hypoxia-reoxygenation injury in cardiomyocytes (1) Extraction and culture of primary cardiomyocytes from Sprague-Dawley (SD) rats Neonatal rat cardiomyocytes (NRCMs) were extracted from 1-day-old SD rats and sterilized with 75% alcohol. The left ventricle was quickly removed using pre-autoclaved ophthalmic scissors, cut into small pieces, and digested with 0.08% trypsin. The isolated cardiomyocytes were incubated for 36 h in DMEM / F12 medium supplemented with 15% FBS for subsequent studies.

[0027] (2) Construction of a primary rat cardiomyocyte hypoxia-reoxygenation model To simulate in vitro ischemia-reperfusion injury, NRCM cells were subjected to hypoxia / reoxygenation (H / R) treatment. First, the cell culture medium was replaced with hypoxia buffer (components: NaCl 118 mM, NaHCO3 24 mM, NaH2PO4·2H2O 1 mM, anhydrous CaCl2 2.5 mM, MgCl2·6H2O 1.2 mM, sodium lactate 20 mM, KCl 16 mM, 2-D-ribose and 2-deoxyglucose 10 mM), and incubated under hypoxic conditions (1% O2) for 6 h. Then, the cells were incubated in DMEM / F12 medium containing 10% FBS and 1% penicillin / streptomycin, and cultured for another 24 h in a cell culture incubator under normal oxygen conditions (21% O2).

[0028] (3) Effects of different concentrations of PC4 on the level of endoplasmic reticulum oxidative stress in primary cardiomyocytes After culturing NRCM in media containing 0.25 μM and 0.5 μM PC4 for 6 h, respectively, an in vitro hypoxia-reoxygenation operation was performed, denoted as H / R + 0.25 μM PC4 and H / R + 0.5 μM PC4, respectively. A model group (H / R) without drug treatment and a blank control group (Control) were also set up.

[0029] The results are as follows Figure 3 As shown, compared with the blank control group and the model group, treatment with 0.25 μM and 0.5 μM PC4 significantly reduced the expression level of GRP78, indicating that PC4 treatment can significantly reduce the endoplasmic reticulum oxidative stress level, and the effect of 0.5 μM PC4 is more obvious.

[0030] (4) Effects of different treatment times on the level of endoplasmic reticulum oxidative stress in primary cardiomyocytes After culturing NRCM in medium containing 0.5 μM PC4 for 6 h, hypoxia-reoxygenation was performed, with reoxygenation times set at 6 h and 24 h, denoted as H / R 6h + PC4 and H / R 24h + PC4, respectively. A model group without drug treatment (H / R 6h and H / R 24h) and a blank control group (Control) were also set up.

[0031] The results are as follows Figure 4 As shown, after drug administration, compared with the control group and 6 hours of hypoxia-reoxygenation, the endoplasmic reticulum oxidative stress level of primary myocardial cells after 24 hours of hypoxia-reoxygenation was significantly reduced.

[0032] Therefore, subsequent experiments used 0.5 μM PC4 administration and the hypoxia-reoxygenation time was selected as 24 h.

[0033] Example 3: CCK8 cell viability assay (1) The inoculation density in the 96-well plate is 2×10⁻⁶. 4 Primary cardiomyocyte suspension (100 μL / well) was prepared by pre-culturing the culture plate in an incubator for 24 h. (2) Add different concentrations (0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 50 and 100 μM) of the test drug to the culture plate; (3) Incubate the culture plate in an incubator for 6 h; (4) Add 10 μL of CCK-8 solution to each well; (5) Place the culture plate in an incubator and incubate for 1-4 hours; (6) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader and calculate the cell viability.

[0034] Cell viability = [(As-Ab) / (Ac-Ab)] × 100% As: Absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution); Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drug); Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drug).

[0035] like Figure 2 As shown in the results, compared with the control group, when the concentration of Phytochelatin 4 was in the range of 0.1 to 0.5 μM, the cell viability remained above 90%, indicating good safety.

[0036] Example 3: Evaluation of a rat primary cardiomyocyte hypoxia-reoxygenation model 1. The drug treatment was divided into four groups, as shown in Table 1.

[0037] Table 1 Experimental Groups 2. Western blot detection of endoplasmic reticulum stress-related proteins After cell sample processing, total protein was lysed in RIPA lysis buffer (Beyotime, China) containing a 1:100 dose of protease inhibitor (Thermo Fisher, USA). Equal amounts of protein (30 μg) were separated using 7.5% or 10% SDS-PAGE gels, then transferred to PVDF membranes (0.22 μm, Millipore, USA), blocked with 5% skim milk at room temperature for 1 h, washed with TBST, and incubated overnight at 4°C with the primary antibody. The following day, the membrane was washed three times with TBST for 5 min each time, then incubated with HRP-labeled secondary antibody at room temperature for 1 h, washed three times with TBST, developed using ECL chemiluminescence, and photographed using a gel imaging system. Grayscale analysis was performed using GAPDH as an internal control. Antibodies against the following proteins were used as primary antibodies: ATF6 (Proteintech, China); p-IRE1α, IRE1α, CHOP, GAPDH (Cell Signaling Technology, USA); IMPAD1 (Boster Biological, China); and ubiquitin (Ub, PTM-BIO, China). All antibodies were diluted according to the manufacturer's recommended ratio before use.

[0038] Test results as follows Figure 5 As shown, after administration, the level of endoplasmic reticulum oxidative stress in primary myocardial cells of ischemia-reperfusion rats was reduced compared with the control group.

[0039] 3. Fluorescent calcium ion detection The calcium concentration in neonatal rat cardiomyocytes (NRCMs) was determined using the Fluo-4 AM calcium ion assay kit (S1061S, Beyotime, China). Cells were incubated with Fluo-4 in HBSS buffer at 37°C for 30 min according to the manufacturer's instructions.

[0040] like Figure 6 As shown in the results, the calcium ion level in primary myocardial cells after hypoxia-reoxygenation decreased compared with the control group after drug administration.

[0041] 4. TUNEL staining of primary cardiomyocytes ① Washing and fixation: The processed cell slides (n=3 / group) need to be washed twice with PBS. After washing, add 0.5 mL of anhydrous methanol to each well and fix at room temperature for 10 min, then wash with PBS 3 times for 5 min each time.

[0042] ② Perforation: Prepare 0.2% Triton-X100 cell permeation solution by weighing 0.1 g BSA and 20 μL Triton-X100 and dissolving them in 10 mL PBS. Add 100 μL of perforation solution to each well, incubate at room temperature for 105 min, and then wash with PBS 3 times for 5 min each time.

[0043] ③ Staining: Add 80 μL of TUNEL working solution in the dark, and incubate the cell culture plate in a 37°C water bath for 1 h. After washing with PBS, add 80 μL of DAPI to each well and incubate at room temperature for 20 min. Wash with PBS 3 times for 5 min each time.

[0044] ④ Mounting: Place the slide in a humidified chamber, add 10 μL of anti-fluorescence quenching mounting medium to the slide, and slowly place the cell smear onto the slide.

[0045] ⑤ Imaging and Result Analysis: Images were captured using an upright fluorescence microscope. Three fluorescence channels were adjusted: red (excitation wavelength 633 nm, apoptotic cells) and blue (excitation wavelength 345 nm, DAPI). Images from all three channels were observed and overlaid at 20x magnification. Three fields of view were randomly selected from each sample for imaging. The ratio of the number of apoptotic cells (purple-pink) to the total number of cell nuclei (blue) after overlay was measured and calculated using ImageJ software. The results were then normalized and statistically analyzed.

[0046] like Figure 7 As shown, TUNEL fluorescence staining results of four groups of primary cardiomyocytes indicated that, compared with the control group, apoptosis of hypoxic-reoxygenated primary cardiomyocytes was reduced after drug administration.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of Phytochelatin 4 in the preparation of drugs for myocardial hypoxia-reoxygenation injury.

2. Application of Phytochelatin 4 in the preparation of drugs for the prevention and treatment of myocardial ischemia-reperfusion injury.

3. The application as described in claim 1 or 2, characterized in that, Phytochelatin 4 achieves its effect of resisting myocardial hypoxia-reoxygenation injury or preventing myocardial ischemia-reperfusion injury by inhibiting cardiomyocyte apoptosis.

4. The application as described in claim 1 or 2, characterized in that, Phytochelatin 4 achieves its effect of resisting myocardial hypoxia-reoxygenation injury or preventing myocardial ischemia-reperfusion injury by alleviating myocardial cell calcium overload induced by hypoxia-reoxygenation.

5. The application as described in claim 1 or 2, characterized in that, Phytochelatin 4 achieves its effect of resisting myocardial hypoxia-reoxygenation injury or preventing myocardial ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress in cardiomyocytes.

6. The application as described in claim 5, characterized in that, Phytochelatin 4 inhibits endoplasmic reticulum stress by downregulating the expression of endoplasmic reticulum stress markers GRP78, ATF6, p-IRE1α, and / or CHOP.

7. The application as described in claim 1, characterized in that, Phytochelatin 4 achieves its effect of resisting myocardial hypoxia-reoxygenation injury or preventing myocardial ischemia-reperfusion injury by reducing the expression level of GRP78.

8. A drug for preventing and treating myocardial ischemia-reperfusion injury, characterized in that, The active ingredient contains Phytochelatin 4.

9. The medicament as described in claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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