Preparation and application of myocardial cell protection preparation by recombining drug containing oxidoreductase-like domain 1 protein-OXLD1 protein

Abstract

The invention discloses preparation and application of a recombined medicine containing oxidoreductase-like domain 1 protein-OXLD1 protein to a myocardial cell protection preparation, OXLD1 protein expression-increased myocardial cells can reduce the injury effect of hypoxia-induced myocardial cells, reduce the mitochondrial injury of the myocardial cells and improve the survival rate of the myocardial cells, and the OXLD1 protein expression-increased myocardial cells can be reduced. Under the condition that the OXLD1 has the beneficial effects, the OXLD1 is non-toxic to cells and does not influence the cell state; the OXLD1 is used for inhibiting mitochondrial damage of hypoxia-induced H9c2 myocardial cells; the OXLD1 promotes the activity of ETC complexes I and V and improves the activity of electron acceptors, and ATP generated in the OXPHOS process is increased at the same time. According to the present invention, the OXLD1 level is inhibited under the hypoxia, the mitochondrial membrane potential (MMP) can be reduced by inhibiting the OXLD1, the reactive oxygen species (ROS) generation can be increased, the mitochondrial damage under the normal oxygen and hypoxia conditions can be aggravated, the OXLD1 can be adopted as the hypoxia damage protection factor by maintaining the mitochondrial function, and the good application prospects are provided in the myocardial protection, and the myocardial infarction prevention and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of cardiovascular disease drugs, and relates to myocardial protective drugs and applications thereof, and specifically to the preparation of a myocardial cell protective preparation containing a recombinant oxidoreductase-like domain 1 protein-OXLD1 protein drug and its application. Background Art

[0002] Myocardial infarction is a cardiovascular disease with a high incidence rate, mainly due to myocardial necrosis caused by acute persistent coronary ischemia and hypoxia. So far, there is no specific drug that can effectively treat myocardial damage after myocardial infarction. In addition, in the treatment of myocardial infarction, reperfusion therapy for acute myocardial infarction can cause adverse reactions such as ischemia-reperfusion injury, in which mitochondria play an important role. Mitochondria, composed of more than 1,200 proteins, are important for many cellular processes. Its dysfunction can lead to hundreds of metabolic diseases, and any disorder of mitochondrial-related proteins can have a profound impact on cells. However, the role of some of these proteins remains unknown. A new protein, Oxidoreductase Like Domain Containing 1 (OXLD1), has attracted attention due to its association with mitochondria. OXLD1 is located in the terminal region of 17q25.3. Some studies have found that the gene located in the terminal region of 17q25.3 is considered to be a complex I assembly factor involved in a variety of mitochondrial processes, including respiratory chain work. The terminal region of 17q25.3 is considered to be a new cardiac susceptibility site. In addition, the researchers also found a possible association between OXLD1 and the key mitochondrial regulatory protein c7orf55. These evidences provide a basis for studying OXLD1. The 17q25.3 terminal region is considered to be a new susceptibility site for cardiac dysfunction.

[0003] The heart is necessary for blood circulation and oxygen delivery to the whole body. At the same time, the heart has an intrinsic oxygen demand to maintain effective contractility. To explore the relevance of OXLD1 to cardiac dysfunction, we examined the relationship between OXLD1 and mitochondrial energy metabolism. Total ATP is produced through mitochondrial oxidative phosphorylation and glycolysis, which are the two major metabolic pathways for the production of ATP in mammalian cells. Is the effect of OXLD1 expression on the rate of ATP production due to oxidative phosphorylation or glycolysis? To address this question, we measured OXPHOS and glycolysis. Our experimental data suggest that OXLD1 improves cardiomyocyte respiratory function through OXPHOS rather than glycolysis. The OXPHOS system is responsible for cellular energy conversion, and its deficiency may lead to mitochondrial diseases. Mitochondria are the center of aerobic respiration. In fact, more than 90% of ATP is produced by OXPHOS in mitochondria. When evaluating the effect of OXLD1 on the enzyme activities of mitochondrial complexes, we found that complexes I and V showed obvious changes. Complex I is the mitochondrial respiratory function regulating machinery, the main site of ROS production, and is involved in apoptosis. Previous studies have shown that the 17q25.3 gene is involved in mitochondrial processes, including multiple respiratory chain functions, as a complex I assembly factor.

[0004] So far, there is no report on the application of OXLD1 protein in myocardial protection or treatment of related diseases, and there is no myocardial protective drug with OXLD1 as the main component. The present invention aims to verify the myocardial protective effect of OXLD1 and the prevention and treatment of myocardial infarction, thereby providing a new myocardial protective drug. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, and to provide the preparation and application of OXLD1 protein as a drug for protecting myocardial cells, preventing myocardial infarction, and increasing cardiac function. The research of the present invention found that OXLD1 promoted the activity of ETC complexes I and V, thereby increasing the activity of electron accepting receptors, and at the same time, the ATP generated by the OXPHOS process increased. In addition, we found that the level of OXLD1 was inhibited under hypoxia; inhibition of OXLD1 can reduce mitochondrial membrane potential (MMP) and increase the generation of reactive oxygen species (ROS), thereby aggravating mitochondrial damage under normoxic and hypoxic conditions. Our research results show that OXLD1 acts as a protective factor for myocardial hypoxic damage by maintaining mitochondrial function, and has a good application prospect in protecting the myocardium, preventing and treating myocardial infarction.

[0006] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0007] The preparation of a myocardial cell protective preparation containing a recombinant oxidoreductase-like domain 1 protein-OXLD1 protein drug comprises the following steps: selecting a specific segment according to the OXLD1 gene sequence to prepare a recombinant plasmid, and constructing a pET-B2M subclone expression vector. The recombinant protein was expressed by Escherichia coli prokaryotes; the recombinant protein was expected to express a size of 16 kDa; 100 μL of the strain stored at -20 ℃ was inoculated into 100 mL LB liquid medium and cultured overnight with shaking; 100 mL of the bacterial solution was inoculated into 2000 mL LB liquid medium, and cultured at 37 ℃ until OD600 was about 0.6, and the culture temperature was lowered to 30 ℃; IPTG inducer was added to a final concentration of 0.5 mM, and the culture was continued with shaking at 30 ℃ for 3-4 h; the bacteria were collected by centrifugation at 8000 rpm for 3 min, resuspended in 50 mL pre-cooled NTA-0 buffer, and ice bathed for 30 min; the bacteria were ultrasonically disrupted, and the parameters were set to power 200 W, work 3 s, pause 4 s, and time 25-30 min; centrifuged at 16000 rpm at 4 ℃ for 50 min, and the supernatant and precipitate were collected; a small amount of supernatant and precipitate were taken for SDS-PAGE detection, and the remaining supernatant and precipitate were collected for 4 ℃ for use, and the recombinant protein-OXLD1 drug is obtained.

[0008] In the present invention, OXLD1 is subcellularly located in mitochondria and cytoplasm.

[0009] In the present invention, under hypoxic conditions, myocardial cells are damaged and the expression of the protective protein OXLD1 is inhibited.

[0010] In the present invention, OXLD1 can alleviate mitochondrial damage caused by the decrease of MMP due to hypoxia, thereby protecting the energy metabolism of myocardial cells.

[0011] In the present invention, the expression of OXLD1 protein inhibits the mitochondrial damage of H9c2 cardiomyocytes induced by hypoxia.

[0012] In the present invention, OXLD1 protein is co-localized with mitochondria.

[0013] In the present invention, OXLD1 can help protect damaged cells by affecting the respiration of H9c2 cardiomyocytes.

[0014] In the present invention, the increase of mitochondrial respiratory function by OXLD1 is achieved through mitochondrial complexes I and V.

[0015] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art.

[0016] Cardiomyocytes with increased OXLD1 protein expression can reduce hypoxia-induced cardiomyocyte injury, reduce cardiomyocyte mitochondrial damage, and improve cardiomyocyte survival. In addition, OXLD1 itself is non-toxic to cells and does not affect cell status when it can have a beneficial effect. OXLD1 protects cardiomyocytes by inhibiting hypoxia-induced mitochondrial damage in H9c2 cardiomyocytes and by affecting the energy metabolism of H9c2 cardiomyocytes. In addition, our data show that OXLD1 promotes the activity of ETC complexes I and V, thereby increasing the activity of electron acceptors. At the same time, the ATP produced by the OXPHOS process increases. In addition, we found that the level of OXLD1 was inhibited under hypoxia; inhibition of OXLD1 can reduce mitochondrial membrane potential (MMP) and increase reactive oxygen species (ROS) production, thereby aggravating mitochondrial damage under normoxic and hypoxic conditions. Our results show that OXLD1 acts as a protective factor against hypoxic injury by maintaining mitochondrial function, and it has a good application prospect in protecting the myocardium, preventing and treating myocardial infarction.

[0017] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are part of this application and are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings described below are only some embodiments. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0019] Figure 1 The prediction of the subcellular localization of OXLD1 in Example 2 of the present invention was studied, and the data came from the GeneCards website, wherein the localization and confidence of OXLD1 in mitochondria and cytoplasm were obtained.

[0020] Figure 2 This is a graph showing the expression level of OXLD1 in myocardial cells under hypoxic conditions according to Example 3 of the present invention; wherein,

[0021] a: OXLD1 mRNA expression level in cells of each group;

[0022] b: Expression level of OXLD1 protein in cells of each group.

[0023] Figure 3 This is a graph showing the protective detection results of OXLD1 in Example 4 of the present invention on the mitochondrial membrane potential of H9c2 cardiomyocytes induced by hypoxia; wherein,

[0024] a: Fluorescence staining of mitochondrial membrane potential of cells in each group;

[0025] b: Statistical graph of red fluorescence intensity of cells in each group.

[0026] Figure 4 This is a graph showing the detection results of ROS in the protection of OXLD1 from hypoxia-induced mitochondrial damage in H9c2 cardiomyocytes according to Example 5 of the present invention; wherein,

[0027] a+c: ROS staining results of cells in each group detected by flow cytometry;

[0028] b+d: Statistical graphs of the proportion of ROS red fluorescence positive cells in each group.

[0029] Figure 5 This Example 6 is a graph showing the detection results of OXLD1 in the mitochondrial subcellular localization of cardiomyocytes.

[0030] Figure 6 This Example 7 is a result diagram of the effect of OXLD1 on the oxygen consumption rate of H9c2 cardiomyocytes; wherein,

[0031] a: Real-time ATP rate test of cells in each group;

[0032] b: Cell mitochondrial stress test of each group of cells, including ATP production, maximum respiration, proton leak, spare respiration and basal respiration;

[0033] c: Determination of glycolysis rate of cells in each group, including basal glycolysis and compensatory glycolysis.

[0034] Figure 7 This is a graph showing the results of the detection of the effect of OXLD1 on the activity of mitochondrial complexes in Example 8 of the present invention.

[0035] a: Detection results of mitochondrial complex I activity of cardiomyocytes in each group;

[0036] b: Results of the detection of mitochondrial complex II / III activity in myocardial cells of each group;

[0037] c: Detection results of mitochondrial complex IV activity of cardiomyocytes in each group;

[0038] d: Detection results of mitochondrial complex V activity of cardiomyocytes in each group.

[0039] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but are intended to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0041] If the specific conditions are not specified in the specific implementation mode, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased from the market.

[0042] The cardiomyocyte reactive oxygen species detection ROS kit in the specific implementation method was purchased from Sigma-Aldrich Corporation, with the catalog number MAK145; the hippocampus real-time ATP rate test kit was purchased from Agilent, with the catalog number 103592-100; the hippocampus mitochondrial stress test was purchased from Agilent, with the catalog number 103015-100; the hippocampus glycolysis rate test was purchased from Agilent, with the catalog number 103344-100; the mitochondrial complex activity test, the mitochondrial complex I activity kit was purchased from Abcam, with the catalog number ab109721, and the complex II, III, IV, and V activity kits were all purchased from Cayman, with the catalog numbers 700950, 700990, and 701000, respectively.

[0043] All experiments in the specific implementation were repeated at least 3 times independently, and the data were statistically analyzed using Graph Pad Prism 8.0, and the results were expressed as mean ± standard deviation. The t test was used for comparison between two groups; one-way analysis of variance was used for comparison between multiple groups, and Dunnett's test was used for multiple comparisons between two groups. P < 0.05 indicated that the difference was statistically significant.

[0044] Example 1

[0045] Recombinant protein-OXLD1 drug preparation

[0046] According to the design of OXLD1 gene sequence, a specific segment was selected to prepare the recombinant plasmid, and the pET-B2M subcloning expression vector was constructed. The recombinant protein was expressed by Escherichia coli prokaryotic cells; the recombinant protein was expected to express a size of 16 kDa. Take 100 μL of the strain stored at -20℃ and inoculate it into 100 mL LB liquid culture medium and shake and culture it overnight; take 100 mL of the bacterial solution and inoculate it into 2000 mL LB liquid culture medium, expand the culture at 37℃ to OD600 of about 0.6, and reduce the culture temperature to 30℃; add IPTG inducer to a final concentration of 0.5 mM, and continue to shake and culture at 30℃ for 3-4 h; collect the bacteria by centrifugation at 8000 rpm for 3 min, resuspend in 50 mL pre-cooled NTA-0 buffer, and ice bath for 30 min; ultrasonically disrupt the bacteria, and the parameters are set to power 200 W, work 3 s, pause 4 s, and time 25-30 min; centrifuge at 16000 rpm at 4℃ for 50 min, collect the supernatant and precipitate; take a small amount of supernatant and precipitate for SDS-PAGE detection, and the remaining supernatant and precipitate are stored at 4℃ for use.

[0047] Example 2

[0048] This study focuses on the subcellular localization of OXLD1, and the data comes from the GeneCards website:

[0049] The results are as follows Figure 1 As shown, Figure 1 Panel A shows that the subcellular localization of OXLD1 is predicted to be mainly in mitochondria and cytoplasm, and the shades of green correspond to the confidence levels of the data, 1-5.

[0050] Example 3

[0051] This example studies the expression level of OXLD1 in cardiomyocytes under hypoxic conditions:

[0052] 1. Cell and reagent preparation:

[0053] H9c2 cardiomyocytes were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin. The normoxic cells were placed in an incubator at 37°C, 20% oxygen, and 5% carbon dioxide; the hypoxic cells were placed in an incubator at 37°C, 1% oxygen, and 5% carbon dioxide. The culture medium was changed every 48 hours, and RNA was extracted using a kit purchased from Thermo Fisher Scientific Inc., catalog number AM1912, when the cell density was 80% to 90%. In addition, RIPA lysis buffer purchased from Beyotime was used to extract protein.

[0054] 2. Experimental groups:

[0055] Blank control group (Control group): T25 cell culture flask, high-glucose DMEM culture, cell density of 80-90%, placed in an incubator at 37°C, 20% oxygen, 5% carbon dioxide for 24 hours;

[0056] Experimental hypoxia group (Hypoxia group): T25 cell culture flask, high-glucose DMEM culture, cell density of 80-90%, placed in 37 ° C, 1% oxygen, 5% carbon dioxide incubator hypoxia culture for 24 hours;

[0057] 3. qPCR was used to detect the expression of OXLD1 gene in cardiomyocytes. RNA was extracted from total RNA using RNAqueous® Total RNA Isolation Kit (Thermo Fisher Scientific Inc, AM1912, Waltham, MA). RT-qPCR was performed using PrimeScriptTM RT reagent Kit (Takara Biomedical Technology Co., Ltd., RR047A, Osaka, Japan) and GoTaq qPCR Master Mix (Promega Corporation, A6001, Madison, WI) in CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., C1000 Touch, Hercules, CA). β-actin was used as an internal reference. The sequences of RT-PCR primers were as follows: OXLD1-F: 5′-GCTTGGAGGAGTCATGGTCA-3′, OXLD1-R: 5′-TTCTGCGGCTATCAGTGGTT-3′, β-actin-F: 5′-AGGCATCCTGACCCTGAAGTAC-3′, and β-actin-R: 5′-GAGGCATACAGGGACAACACAG-3′.

[0058] 4. The expression of OXLD1 protein was detected by WB. The specific RIPA lysis buffer containing Phenylmethanesulfonyl Fluoride (PMSF) (Coolaber, SL1020, Beijing, China) was used to lyse cells and extract proteins. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad Laboratories, Inc., 5671034, Hercules, CA) and transferred to PVDF (ThermoFisher Scientific Inc, 88520, Waltham, MA) membranes. After blocking, the PVDF membrane was incubated with primary antibodies at 4°C overnight. Then, the PVDF membrane was incubated with secondary antibodies at room temperature for 2 hours (1:3000, Abcam, ab6721, Cambridge, England). The primary antibodies used were: OXLD1 (Abcam, ab188306, Cambridge, England) and β-actin (Abcam, ab8226, Cambridge, England). Repeat the experiment 3 times.

[0059] Test results such as Figure 2 As shown, Figure 2 Figure a in the middle shows the OXLD1 mRNA gene expression levels of cells in each group after treatment. Among them, the cells treated with hypoxia in an incubator with 1% oxygen and 5% carbon dioxide for 24 hours showed a significant downregulation of mRNA gene expression compared with the Control group (n = 3, P < 0.01). Figure 2 Figure b is a quantitative statistical chart of the expression levels of OXLD1 protein in the two groups of cardiomyocytes. It can be seen that the cells treated with hypoxia in an incubator with 1% oxygen and 5% carbon dioxide for 24 hours also had a significantly downregulated expression of OXLD1 protein compared with the Control group (n = 3, P < 0.01).

[0060] This shows that under hypoxic conditions, cardiomyocytes are damaged and the expression of the protective protein OXLD1 is inhibited.

[0061] Example 4

[0062] This example studies the protection of OXLD1 in the mitochondrial membrane potential of H9c2 cardiomyocytes induced by hypoxia:

[0063] 1. Cell and reagent preparation

[0064] H9c2 cardiomyocytes were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin. Normoxic cells were placed in an incubator at 37°C, 20% oxygen, and 5% carbon dioxide; hypoxic cells were placed in an incubator at 37°C, 1% oxygen, and 5% carbon dioxide. OXLD1 overexpression (OXLD1 OE) plasmid construction: OXLD1 [Rattus norvegicus (Norway rat)] (NCBIaccession NO: NM_001305985.1) was cloned into the EcoRI and XbaI sites of the plenti-GII-CMV-CBH-GFP-2A-Puro (Amp) Vector. SiRNA OXLD1 558: Gene interference of OXLD1 was performed by constructing siRNA, the sequence of which is F: GCACGUGACUGAUGAGAACTT; R: GUUCUCAUCAGUCACGUGCTT.

[0065] 2. Experimental groups:

[0066] Normoxia control group: cardiomyocytes H9C2 were cultured in an incubator at 37°C, 20% oxygen, and 5% carbon dioxide for 24 hours;

[0067] Hypoxia group: cardiomyocytes H9C2 were cultured in an incubator at 37°C, 1% oxygen, 5% carbon dioxide for 24 hours;

[0068] OXLD1-overexpression normoxia group (Normoxia OXLD1-OE group): cardiomyocytes H9C2 were transfected with OXLD1 overexpression plasmid and cultured in an incubator at 37°C, 20% oxygen, and 5% carbon dioxide for 24 hours;

[0069] OXLD1 overexpression hypoxia group (Hypoxia OXLD1-OE group): cardiomyocytes H9C2 were transfected with OXLD1 overexpression plasmid and cultured in an incubator at 37°C, 1% oxygen, 5% carbon dioxide for 24 hours;

[0070] OXLD1 knockdown normoxia group (Normoxia OXLD1-KD group): cardiomyocytes H9C2, after being transfected with OXLD1 knockdown plasmid, were cultured in an incubator at 37°C, 20% oxygen, 5% carbon dioxide for 24 hours;

[0071] OXLD1 knockdown hypoxia group (Hypoxia OXLD1-KD group): cardiomyocytes H9C2 were transfected with OXLD1 knockdown plasmid and cultured in an incubator at 37°C, 1% oxygen, 5% carbon dioxide for 24 hours;

[0072] 3. Use laser confocal microscopy to detect mitochondrial membrane potential (MMP). Tetramethylrhodamine ethyl ester (TMRE) fluorescent dye was used to detect MMP. The maximum excitation wavelength of TMRE is 549nm, and the maximum emission wavelength is 574nm. It is red or orange-red under a fluorescent microscope. Cells were planted in confocol special cell culture dishes. The experiment was carried out at a cell density of 60%-70%. The cells were washed twice with PBS, and 400μL of detection working solution was added to each well. Incubated at 37℃ in the dark for 60min. After the incubation, the staining effect was observed under a fluorescence microscope and detected using a laser confocal microscope (Leica, STELLARIS 8, Weizler, Germany). The fluorescence intensity was measured using ImageJ software. The fluorescence intensity can reflect the mitochondrial membrane potential. As the fluorescence intensity decreases, the membrane potential decreases.

[0073] 4. Test results such as Figure 3 As shown, Figure 3 Figure a shows the cell staining (the scale bar is 50 μm). It can be clearly seen that compared with the cells in the Normoxia group, the mitochondrial membrane potential of the Hypoxia group decreased significantly, while the mitochondrial membrane potential of the cells in the OXLD1 overexpression group (Hypoxia OXLD1-OE) did not decrease, but increased; the mitochondrial membrane potential of the OXLD1 knockdown group decreased significantly, both under normoxic and hypoxic conditions. The statistical graph was drawn by the fluorescence intensity statistics of the red fluorescence staining, as shown in Figure 2. Figure 3 As shown in Figure b, the OXLD1-OE group significantly increased the mitochondrial membrane potential of cardiomyocytes compared with the control and OXLD-KD groups ( ** P < 0.01 vs. Normoxia, ## P < 0.01 vs. Hypoxia).

[0074] The MMP test results showed that OXLD1 could alleviate mitochondrial damage caused by hypoxia-induced MMP decrease, thereby protecting myocardial cell energy metabolism.

[0075] Example 5

[0076] This example studies the protective effect of OXLD1 on mitochondrial damage in H9c2 cardiomyocytes induced by hypoxia:

[0077] 1. Cells and reagents were prepared as described in Example 3.

[0078] 2. Experimental grouping refers to Example 3.

[0079] ROS content was detected by flow cytometry. The intracellular ROS kit (Sigma-Aldrich Corporation, MAK145, St. Louis, MO) was used to detect the ROS content of H9c2 cells. After ROS staining with the probe, the cells were collected and the ROS content was detected by flow cytometry. Reactive oxygen species (ROS) refers to oxygen-containing atoms or atomic groups with active chemical properties, including superoxide anion free radicals, hydrogen peroxide (H2O2), singlet oxygen, hydroxyl free radicals (HO.), alkane peroxide free radicals, lipid peroxide free radicals, etc. They are involved in physiological and pathological processes such as cell growth and proliferation, development and differentiation, aging and apoptosis. The dihydroethidium (DHE) reactive oxygen species detection kit uses the fluorescent probe DHE for reactive oxygen species detection. DHE is an indicator of peroxides, which can freely penetrate the living cell membrane into the cell, show blue fluorescence in the cytoplasm, and be oxidized by intracellular ROS to form oxyethidium; oxyethidium can be incorporated into chromosomal DNA, making the cell nucleus show bright red fluorescence. Based on the generation of red fluorescence in living cells, the amount and changes of cellular ROS content can be determined. DHE is mainly oxidized by superoxide anion ROS in cells, which can be directly observed by flow cytometry or fluorescence microscopy. It is a quick and easy classic method for detecting ROS in tissues or cultured living cells. At an excitation wavelength of 535nm and an emission wavelength of around 610nm, the amount and changes of cellular ROS content can be determined based on the generation of red fluorescence in living cells in a suitable box. It can be directly observed by flow cytometry or fluorescence microscopy. It is a quick and easy classic method for detecting ROS in tissues or cultured living cells. H9C2 is an adherent cell. The cells were rinsed twice with PBS and then digested with trypsin. The cells were resuspended in cold PBS (5×10 5 -1×10 6 The dye solution was diluted 500 times with fresh culture medium to the required concentration (50 μM) and incubated at room temperature or 37°C in the dark for 60 minutes. ROS-negative cells have only very low fluorescence intensity, while ROS-positive cells have strong red fluorescence.

[0080] 2. Test results such as Figure 4 As shown, Figure 4 Figure a+c in the figure shows the results of flow cytometry detection of cell ROS staining. The red fluorescence positive cells are counted and a statistical graph is drawn as shown in Figure 4As shown in Figures b+d, the OXLD1-OE group significantly reduced the amount of ROS compared with the control group (**P<0.01), and the OXLD1-KD group significantly increased the amount of ROS compared with the control group (**P<0.01). These results indicate that the expression of OXLD1 protein inhibits hypoxia-induced mitochondrial damage in H9c2 cardiomyocytes.

[0081] Example 6

[0082] This example studies the mitochondrial subcellular localization of OXLD1 in cardiomyocytes:

[0083] 1. Cell and reagent preparation

[0084] H9c2 cardiomyocytes were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 mg / mL streptomycin. The normoxic cells were placed in an incubator at 37°C, 20% oxygen, and 5% carbon dioxide.

[0085] 2. Colocalization of OXLD1 and mitochondria. H9C2 cells were plated in confocol dishes, washed twice with PBS, and fixed with 4% paraformaldehyde for 30 minutes at room temperature. Washed three times with PBS containing 0.1% Triton X-100 for 5 minutes each time. After blocking, incubated with 1-5% BSA and 0.1% Triton X-100 in PBS at a dilution ratio of 1:100 with OXLD1 antibody (Abcam, ab188306, Cambridge, England) at 4°C overnight. Then, cells were incubated with fluorescent secondary antibody for 2 hours at room temperature (Alexa Fluor®488, Abcam, ab150077, Cambridge, England). After washing, cells were incubated with anti-Tom20 antibody (CoraLite®594-conjugated, Proteintech Group, Inc., CL594-11802, Chicago, IL) for 2 hours at room temperature, and then the nuclei were stained. The images were taken under a laser confocal microscope (Leica, STELLARIS 8, Wetzlar, Germany).

[0086] 3. Test results such as Figure 5 As shown, we used DAPI to label the nuclei of H9C2 cells (blue fluorescence), Tom20 as a marker protein for mitochondria (red fluorescence), and OXLD1 protein with green fluorescence. The orange particles indicate the fusion between mitochondria and OXLD1. Bar = 50 μm. We detected the enrichment of OXLD1 in mitochondria, and we noticed that OXLD1 protein colocalized with mitochondria.

[0087] Example 7

[0088] This example studies the effect of OXLD1 on the oxygen consumption rate of H9c2 cardiomyocytes:

[0089] 1. Cells and reagents were prepared as described in Example 3.

[0090] 2. Experimental grouping refers to Example 3.

[0091] 3. The total ATP production rate of OXLD1-OE and OXLD1-KD cells under normoxic and hypoxic conditions was measured by the Agilent Seahorse Real-Time ATP Rate Assay. The rate of cellular adenosine triphosphate (ATP) production is a highly informative measurement describing cellular metabolism, as ATP is the primary energy currency ubiquitously present in cells. Metabolic regulation of cells allows cells to adjust changes in ATP production in response to changes in ATP demand to maintain total intracellular ATP levels. The Agilent Seahorse XF Real-Time ATP Rate Assay is designed to measure the total ATP production rate in living cells. More importantly, this assay is able to distinguish between mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic ATP production, two major metabolic pathways for ATP production in mammalian cells. The Seahorse XF Real-Time ATP Rate Assay provides a new dynamic and quantitative perspective on cellular bioenergetics by providing real-time measurements of cellular ATP production rates and quantitative phenotypes of cellular energy balance. The Seahorse Real-Time ATP Rate Assay measures the real-time extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of cells directly in the Seahorse analyzer after the sequential addition of mitochondrial inhibitors (oligomycin and rotenone / antimycin A, Rote / AA) - indicators of two major energy production pathways: glycolysis and oxidative phosphorylation. After analysis, cells in each well are counted and results are normalized to values ​​per 5,000 cells. Calculation of mitochondrial and glycolytic ATP production rates provides a real-time measure of cellular ATP production and a quantitative phenotype of cellular energy balance.

[0092] The ATP production rate related to lactate synthesis (expressed in pmol ATP / min); mitoATP production rate (mitoATPProduction Rate): the ATP production rate related to mitochondrial oxidative phosphorylation (expressed in pmol ATP / min); the total ATP production rate (Total ATP Production Rate): the sum of the mitochondrial ATP production rate and the glycolysis ATP production rate of living cells under appropriate experimental conditions.

[0093] The Agilent Seahorse XF Cell Mitochondrial Stress Test characterizes key parameters of mitochondrial function by measuring the oxygen consumption rate (OCR) of cells directly on the Seahorse XFe and XF Extracellular Flux Analyzers. It is a plate-based live cell assay that monitors the OCR in real time. The Agilent Seahorse Mitochondrial Stress Test measures key parameters of mitochondrial function by measuring the OCR of cells directly. Compounds oligomycin, FCCP, Rote / AA are injected sequentially to measure ATP production, maximal respiration, and non-mitochondrial respiration, respectively. These parameters are then used in conjunction with basal respiration to calculate proton leak and spare respiratory capacity. After analysis, cells are counted in each well and the results are normalized to values ​​per 5,000 cells. Basal respiration: Oxygen consumption used to meet cellular ATP demand and mitochondrial proton leak. Represents the energy demand of the cell under basal conditions; ATP Production: The oxygen consumption rate that is reduced by the addition of the ATP synthase inhibitor oligomycin, representing the portion of basal respiration that is used to drive ATP production. Represents mitochondrial ATP production used to meet the energy needs of the cell; H+ (Proton) leak: The remaining part of basal respiration that is not coupled to ATP production. Proton leak can be seen as a marker of mitochondrial damage or as a mechanism to regulate mitochondrial ATP production; Maximal respiration: The maximum oxygen consumption rate obtained after the addition of the uncoupler FCCP. FCCP simulates physiological "energy demand" by stimulating the respiratory chain to operate at maximum capacity, resulting in rapid oxidation of substrates (sugars, fats, and amino acids) to meet this metabolic challenge. Shows the maximum respiratory rate that the cell can achieve; Spare respiratory capacity: This measurement indicates the ability of the cell to cope with energy demands and the gap between cellular respiration and its theoretical maximum. The ability of the cell to cope with energy demands can be used as an indicator of cell adaptability or flexibility; Nonmitochondrial respiration: The oxygen consumption that remains after the addition of rotenone and antimycin A because enzymes in a part of the cell continue to consume oxygen. This is important for accurate measurement of mitochondrial respiration.

[0094] The Agilent Seahorse XF Glycolytic Rate Assay is an accurate and reliable assay for measuring cellular glycolysis. The Seahorse XF Glycolytic Rate Assay accurately measures basal glycolytic rates and compensatory glycolytic rates following mitochondrial inhibition. The resulting glycolytic rates are directly comparable to lactate accumulation data by calculating and subtracting the contribution of mitochondrial / TCA cycle derived CO2 to extracellular acidification. Continuous injections of the compounds Rot / AA, 2-deoxy-D-glucose, 2-DG are used to measure cellular glycolytic proton efflux rates. The number of cells per well is counted and the results are normalized to values ​​per 5,000 cells. Glycolysis: For the Seahorse XF Glycolysis Rate Assay, this refers to the conversion of glucose to lactate. Buffer Factor (BF): The buffering capacity of the measurement system, including the assay solution and XF assay conditions (instrument, sensor, and assay consumables). Proton Efflux Rate (PER): The number of protons released by cells into the assay solution over a period of time, expressed in pmol / min. Glycolytic Proton Efflux Rate (glycoPER): The proton efflux rate from glycolysis (minus the effects of CO2 acidification).

[0095] 4. Test results such as Figure 6 As shown in Figure 2, compared with the control group, the ATP generation rate in the OXLD1-OE group was significantly increased under normoxic and hypoxic conditions, while the ATP generation rate in the OXLD1-KD group was significantly decreased ( Figure 6 a). Oxidative phosphorylation (OXPHOS) of OXLD1-OE and OXLD1-KD cells under hypoxia and normoxia was measured by the Agilent hippocampal cell mitochondrial stress test, and glycolysis was measured by the Agilent hippocampal glycolysis rate assay. Compared with the control group, we observed a significant increase in OXPHOS in the OXLD1-KD group, and a significant decrease in OXPHOS in the OXLD1-KD group under normoxia and hypoxia ( Figure 6 b). There was no significant change in glycolysis between the OXLD1-OE group and the OXLD1-KD group ( Figure 6 c). These data indicate that OXLD1 can improve the mitochondrial respiratory function of cardiomyocytes under hypoxia. n = 10. Data are expressed as mean ± SD; compared with the normoxic group, *P < 0.05, **P < 0.01; compared with the hypoxic group, #P < 0.05, ##P < 0.01, the difference was statistically significant. The results of the hippocampal test were normalized to the value per 5000 cells.

[0096] These results suggest that OXLD1 can help protect damaged cells by affecting the respiration of H9c2 cardiomyocytes.

[0097] Example 8

[0098] This example studies the effect of OXLD1 on the activity of mitochondrial complexes:

[0099] 1. Cells and reagents were prepared as described in Example 3.

[0100] 2. Experimental grouping refers to Example 3.

[0101] 3. The activity of mitochondrial complex I was detected by Abcam kit (No. ab109721), and the activities of complexes II to V were detected by Cayman kits (No. 700950, 700990, and 701000), respectively. The activity of the complex was calculated by dividing the change in absorbance over time within the linear range of the reading. We evaluated the effect of OXLD1 on the enzyme activity of mitochondrial complexes I-V. Compared with the control group, the activities of mitochondrial complexes I and V in the OXLD1-OE group were significantly increased under normoxic and hypoxic conditions. The activities of mitochondrial complexes I and V in the OXLD1-KD group were significantly decreased under normoxic and hypoxic conditions ( Figure 7 a, Figure 7 d). However, there were no significant changes in mitochondrial complexes II / III and IV ( Figure 7 bc). Our results suggest that the increase in mitochondrial respiratory function by OXLD1 is achieved through mitochondrial complexes I and V.

[0102] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. Preparation of a myocardial cell protective preparation containing a recombinant oxidoreductase-like domain 1 protein-OXLD1 protein drug, characterized in that: The method comprises the following steps: selecting a specific segment according to the OXLD1 gene sequence to prepare a recombinant plasmid, and constructing a pET-B2M subclone expression vector. The recombinant protein was expressed by Escherichia coli prokaryotes; the recombinant protein was expected to be expressed in an amount of 16 kDa; 100 μL of the strain stored at -20 ℃ was inoculated into 100 mL of LB liquid medium and cultured overnight with shaking; 100 mL of the bacterial solution was inoculated into 2000 mL of LB liquid medium, cultured at 37 ℃ until OD600 was about 0.6, and the culture temperature was lowered to 30 ℃; IPTG inducer was added to a final concentration of 0.5 mM, and cultured at 30 ℃ for 3-4 h; the bacteria were collected by centrifugation at 8000 rpm for 3 min, resuspended in 50 mL of pre-cooled NTA-0 buffer, and placed on ice for 30 min; the bacteria were broken by ultrasonication, with the parameters set to 200 W power, 3 s working, 4 s pause, and 25-30 min time; centrifuged at 16000 rpm at 4 ℃ for 50 min, and the supernatant and precipitate were collected; a small amount of supernatant and precipitate were taken for SDS-PAGE The remaining supernatant and precipitate were stored at 4°C for later use, and the recombinant protein-OXLD1 drug was obtained.

2. Application of a recombinant drug containing oxidoreductase-like domain 1 protein - OXLD1 protein to a myocardial cell protection preparation, characterized in that: OXLD1 subcellular localization is in mitochondria and cytoplasm.

3. Application of a recombinant OXLD1 protein drug to a myocardial cell protection preparation, characterized in that: Under hypoxic conditions, cardiomyocytes are damaged and the expression of the protective protein OXLD1 is inhibited.

4. Application of a recombinant OXLD1 protein drug to a myocardial cell protection preparation, characterized in that: OXLD1 can alleviate mitochondrial damage caused by hypoxia-induced MMP decrease, thereby protecting cardiomyocyte energy metabolism.

5. Application of a recombinant OXLD1 protein drug to a myocardial cell protection preparation, characterized in that: Expression of OXLD1 protein inhibited hypoxia-induced mitochondrial damage in H9c2 cardiomyocytes.

6. Application of a recombinant OXLD1 protein drug to a myocardial cell protection preparation, characterized in that: OXLD1 protein colocalizes with mitochondria.

7. Application of a recombinant drug containing oxidoreductase-like domain 1 protein - OXLD1 protein to a myocardial cell protection preparation, characterized in that: OXLD1 may help protect damaged H9c2 cardiomyocytes by affecting their respiration.

8. Application of a recombinant OXLD1 protein drug to a myocardial cell protection preparation, characterized in that: OXLD1 increases mitochondrial respiratory function through mitochondrial complexes I and V.

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