Application of AQP1 in preparation of medicine for treating hyperinsulinemia myocardial injury

Abstract

The invention provides an application of AQP1 in preparation of a medicine for treating hyperinsulinemia myocardial injury, and relates to the field of biomedicine.The AQP1 protein serves as a target spot and is inhibited by using a related inhibitor, so that myocardial edema is effectively reduced, ion homeostasis unbalance is improved, mitochondrial dysfunction is repaired, and the effect of treating hyperinsulinemia myocardial injury is achieved. Therefore, the effect of treating the hyperinsulinemia myocardial injury is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of AQP1 in preparing a drug for treating hyperinsulinemia myocardial injury. Background Art

[0002] Diabetic cardiomyopathy (DCM) is a significant complication of type 2 diabetes mellitus (T2DM), significantly increasing the risk of heart failure and mortality in diabetic patients. DCM is a specific cardiomyopathy independent of other cardiovascular diseases, such as hypertension, coronary artery disease, valvular disease, and congenital heart disease. Clinically, DCM presents with early diastolic dysfunction, late systolic dysfunction, and heart failure. Many metabolic processes associated with diabetes promote myocardial damage and cardiomyocyte death. It is currently believed that insulin resistance and hyperinsulinemia are the primary triggers of DCM in T2DM. Hyperinsulinemia can mediate myocardial fibrosis and left ventricular remodeling, leading to increased ventricular wall stiffness and decreased compliance, inducing early diastolic myocardial dysfunction. Numerous studies have demonstrated that myocardial insulin resistance and hyperinsulinemia are closely linked to sodium and calcium homeostasis in cells. Ion homeostasis in cardiomyocytes is regulated by ion pumps on the cell membrane, and sodium and calcium ions are key factors in regulating these pumps and myocardial function. Abnormal insulin signaling in cardiomyocytes disrupts sodium and calcium homeostasis by affecting the expression of ion pumps. When cells are overloaded with sodium and calcium ions and mitochondrial calcium content decreases, mitochondrial dysfunction is mediated, oxidative stress increases, and ultimately leads to cell death. Sodium ions and water molecules in the human body maintain a dynamic balance. When the sodium ion content in cells increases, water molecules enter the cells through free diffusion, channel transport, and other methods, forming cellular edema. Water balance plays an important role in maintaining myocardial cell function. Edema affects the contractile function of the myocardium by increasing ventricular wall stiffness and reducing ventricular wall compliance; and edema affects the conduction of ventricular electrical signals by affecting the ionic composition of myocardial cells, thereby causing arrhythmias. It can be seen that myocardial water balance is crucial to maintaining normal heart function.

[0003] AQPs (aquaporins, AQPs) are a class of molecular channel proteins that are crucial for maintaining water homeostasis in the myocardium. In 1968, Benga et al. discovered these small proteins involved in water transport and subsequently named them aquaporins (AQPs). Their expression significantly increases the water permeability of cell membranes. AQPs are widely distributed throughout organs, mediating the transport of small molecules such as water, O₂, H₂O, and N₂. Currently, 13 AQP isoforms with a homologous molecular structure have been identified in mammals. They are approximately 320 amino acid residues long, have a molecular weight of approximately 28 kDa, and exist as tetramers on the cell membrane. AQP expression varies across species and organs, with each isoform coordinated to maintain homeostasis. AQP1, AQP4, and AQP7 are the predominant isoforms expressed in the myocardium. AQPs play a crucial role in maintaining myocardial water homeostasis. Water molecules can flow between cells through free diffusion, ion channel transporters, or aquaporins. Under physiological conditions, one-third of the water flowing into the myocardial tissue from the capillaries is regulated by AQPs. Under pathological conditions, AQPs play a major role in regulating water transport. Cardiac function is heavily dependent on water balance. Even a slight increase in water content can impair myocardial contractility. Myocardial edema affects the normal oxidative phosphorylation and ATP synthesis ability of myocardial cells by mediating mitochondrial swelling and increasing the oxygen diffusion distance, thus causing cardiac dysfunction. AQP1 is an important subtype that maintains myocardial water balance. Current studies have found that AQP1 is closely related to many cardiovascular diseases such as myocardial infarction, myocardial ischemia-reperfusion injury, cardiac ischemia-hypoxia, and cardiac extracorporeal circulation surgery. Increased AQP1 expression mediates the occurrence and development of myocardial edema.

[0004] Currently, the mechanism of action and application of AQP1 in hyperinsulinemia-induced myocardial injury are not clear.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides the use of AQP1 in the preparation of a drug for treating hyperinsulinemia myocardial injury, wherein the AQP1 protein is used as a target and is inhibited using relevant inhibitors, thereby effectively improving and reducing myocardial edema, improving ion homeostasis imbalance and repairing mitochondrial dysfunction, thereby playing a role in treating hyperinsulinemia myocardial injury.

[0007] In order to achieve the purpose of the present invention, the present invention provides the following scheme: Application of AQP1 protein as a target in the preparation of drugs for treating hyperinsulinemia-induced myocardial injury.

[0008] Furthermore, the drug exerts its effect by inhibiting the expression level of AQP1 or the activity of AQP1.

[0009] Furthermore, the inhibitor for inhibiting the expression of AQP1 or the activity of AQP1 is any one of Tc-AQP1 or siRNA.

[0010] Furthermore, the inhibitor is siRNA.

[0011] Furthermore, the target sequence of the siRNA is: GCAGCAGAGTAAAGGTCAT.

[0012] Furthermore, the hyperinsulinemia myocardial damage includes myocardial cell edema, sodium and calcium ion overload or mitochondrial dysfunction.

[0013] The present invention also provides a pharmaceutical composition for treating hyperinsulinemia myocardial injury, comprising an AQP1 inhibitor and a pharmaceutically acceptable carrier.

[0014] The present invention also provides an in vitro diagnostic kit for detecting hyperinsulinemia myocardial damage, the kit comprising: a reagent for detecting the expression level of AQP1.

[0015] Furthermore, the reagent includes a specific antibody or nucleic acid probe against AQP1.

[0016] The embodiments of the present invention have the following technical effects: The present invention uses AQP1 protein as a target and further uses siRNA to inhibit AQP1 expression or inhibit AQP1 activity, thereby improving myocardial cell edema, cellular ion homeostasis imbalance and mitochondrial dysfunction induced by high-concentration insulin. Drugs that inhibit AQP1 expression are used to treat hyperinsulinemia myocardial injury, not only providing a new drug target for the prevention or treatment of hyperinsulinemia myocardial injury, but also providing a new approach for the treatment and prevention of this disease and the preparation of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1: Heart tissues of hyperinsulinemia model animals; Figure A shows the fasting blood glucose results between the two groups, Figure B shows the fasting insulin results between the two groups, Figure C shows the HOMA-IR results between the two groups, Figure D shows the weight change trends between the two groups, Figure E shows the OGTT results between the two groups, Figure F shows the ITT results between the two groups, Figure G shows the immunohistochemistry results of HE and AQP1 between the two groups, and Figure H shows the mRNA and protein change trends of AQP1 in heart tissues between the two groups; Figure 2 : Results of high-concentration insulin-induced AQP1 protein expression in cardiomyocytes; Figure A shows the trend of AQP1 protein changes in cardiomyocytes of the two groups; Figure B shows the immunofluorescence staining results of AQP1 in the two groups; Figure C shows the CCK8 results after gradient intervention of different concentrations of insulin in cardiomyocytes; Figures D and E show the trend of NHE1, AKT, p-AKT, and AQP1 protein changes after intervention of different concentrations of insulin; Figure 3 : Results of the effects of AQP1 on water, sodium, and calcium balance and mitochondrial function in cardiomyocytes under high-insulin conditions; Figure A shows cellular edema after high-insulin treatment of cardiomyocytes; Figure B shows water and sodium ion metabolism disorders after high-insulin treatment of cardiomyocytes; Figure C shows sodium and calcium ion expression after high-insulin treatment of cardiomyocytes; Figure 4 Results of the study on the effects of AQP1 on water, sodium, and calcium balance and mitochondrial function in cardiomyocytes under high-insulin conditions; Figure A shows the levels of mitochondrial calcium and ROS in cardiomyocytes after high-insulin treatment; Figure B shows changes in key mitochondrial function indicators after high-insulin treatment; Figure C shows changes in JC-1 mitochondrial membrane potential after high-insulin treatment; Figure 5 si-AQP1 can alleviate high-concentration insulin-induced water, sodium-calcium imbalance and mitochondrial dysfunction in cardiomyocytes; Figures A and B show the results of si-AQP1 knockdown sequence verification; Figure C shows the improvement of cell edema after si-AQP1 treatment under high-concentration insulin intervention; Figure 6 si-AQP1 can alleviate the water, sodium, and calcium imbalances and mitochondrial dysfunction in cardiomyocytes induced by high-concentration insulin. Figure A shows the sodium and calcium ion content in cells and mitochondria induced by si-AQP1 under high-concentration insulin treatment. Figure B shows the effect of si-AQP1 on the reduction of changes in mitochondrial membrane potential in cardiomyocytes under high-concentration insulin treatment. Figure 7 :Si-AQP1 alleviates cellular sodium and calcium overload and changes in mitochondrial calcium content under insulin intervention. DETAILED DESCRIPTION

[0019] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0020] In a first aspect, the present invention provides the use of AQP1 protein as a target in the preparation of a drug for treating hyperinsulinemia-induced myocardial injury.

[0021] In some embodiments, the drug acts by inhibiting the expression level of AQP1 or the activity of AQP1.

[0022] In some embodiments, the inhibitor for inhibiting AQP1 expression or AQP1 activity is Tc-AQP1 or siRNA.

[0023] In some embodiments, the inhibitor is siRNA.

[0024] In some embodiments, the target sequence of the siRNA is: GCAGCAGAGTAAAGGTCAT.

[0025] In some embodiments, the hyperinsulinemia myocardial injury includes any one of cardiomyocyte hypertrophy, sodium and calcium ion overload, or mitochondrial dysfunction.

[0026] On the other hand, the present invention also provides a pharmaceutical composition for treating hyperinsulinemia myocardial injury, comprising an AQP1 inhibitor and a pharmaceutically acceptable carrier.

[0027] On the other hand, the present invention also provides an in vitro diagnostic kit for detecting hyperinsulinemia myocardial injury, the kit comprising: a reagent for detecting the expression level of AQP1.

[0028] In some embodiments, the reagent comprises an antibody or a nucleic acid probe specific for AQP1.

[0029] The following is elaborated with reference to specific embodiments: Example 1: Determination of AQP1 protein expression level in cardiac tissue of hyperinsulinemia model animals 1.1 Experimental Procedure 1.1.1 Animal grouping and tissue collection Sixteen 8-week-old WT rats were randomly divided into two groups: a normal control group (NC group, n=8) and a hyperinsulinemia group (HINS group, n=8). The NC group was fed a standard diet, while the HINS group was fed a 60% kcal high-fat diet. After 16 weeks of feeding, the rats in each group were weighed weekly. Every four weeks, the rats were placed in metabolic cages for 24 hours to monitor their metabolic status. Blood was collected from the medial canthus of the rats' orbits, centrifuged, and serum was collected to assess changes in lipid profiles, liver and kidney function, and insulin levels.

[0030] Hyperinsulinemia model criteria: The fasting plasma insulin level of rats is elevated and statistically significant between the two groups. Subsequently, OGTT and ITT tests were performed. The experimental methods are as follows: (1) Oral glucose tolerance test (OGTT) Before sampling, an OGTT experiment was performed to assess the glucose metabolism of rats. The rats were weighed the day before and fasted overnight but not water. On the morning of the second day, the fasting blood glucose of the tail tip of each group of rats was measured and recorded as the value at 0 min. Then, the rats were gavaged with the prepared 30% glucose solution at a concentration of 2 g / kg. The gavage time of each rat was recorded, and the blood glucose of each group of rats was measured at 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min. The curve was drawn and the area under the curve was calculated to compare the two groups.

[0031] (2) Insulin tolerance test (ITT) Before sampling, an ITT experiment was performed to evaluate the islet function of rats. The rats were weighed the day before and the amount of insulin injected into each group of rats was calculated. The rats in each group fasted for 6 hours and had free access to water. 30% glucose water was also prepared to prevent the rats from developing hypoglycemia. At dawn on the second day, the fasting tail tip blood glucose of the rats in each group was tested and recorded as the 0-min value. Rats were injected intraperitoneally with NovoRapid 30 insulin according to their body weight at a dose of 0.5 U / kg. The blood glucose values ​​were measured 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, and 150 minutes after the injection of insulin. The curves were drawn and the area under the curve (AUC) was calculated to compare the differences between the two groups.

[0032] After the hyperinsulinemia model was successfully established, rats were fasted overnight and anesthetized with an intraperitoneal injection of 500 mg / kg tribromoethanol. Beating heart tissue was removed and placed in sterile saline to allow residual beating capacity to drain residual blood from the heart tissue. Tissue was then weighed and the weight was recorded. Apical tissue was removed with a razor blade and fixed in 4% paraformaldehyde. The remaining tissue was placed in an EP tube and stored at -80°C.

[0033] 1.1.2 H&E staining and immunohistochemical staining (1) H&E staining 1) Remove the wax block wrapped in the embedding machine from -20℃, trim the block until the cut surface is flat, and then slice it. Be careful not to curl the slice. The thickness should be 4-5μm. 2) Place the cut tissue slices on a slide and soak them in 42℃ warm water to fully stretch the tissue slices. 3) Dewax the stretched tissue slices by soaking them in xylene twice, each time for 10 minutes. 4) Dehydrate the xylene-soaked tissue slices in anhydrous ethanol for 5 minutes, then dehydrate them in 90%, 80%, and 70% ethanol once each, each time for 5 minutes, and then soak them in ddH2O. 5) Place the hydrated tissue slices on a slide. 6) Rinse the sections in hematoxylin solution for 3-5 minutes; 7) Place the sections in eosin solution for 1 minute; 8) Rinse the sections with ddH2O to remove excess eosin solution, and then dehydrate the sections in a gradient of 80% ethanol for 2 seconds, 90% ethanol for 2 seconds, anhydrous ethanol for 10 seconds, and xylene for 3 minutes; 9) Immediately remove the sections from the xylene solution, add a drop of neutral gum, and cover with a coverslip.

[0034] (2) Immunohistochemical staining 1) Deparaffinization: First, bake the tissue sections at 60°C for 2 hours. Then, place the sections in xylene I solution for 10 minutes, xylene II solution for 10 minutes, anhydrous ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and finally soak in ddH2O. 2) Antigen Retrieval: Prepare a 1x Tris-EDTA solution, place the sections in the solution, and microwave on high for 5 minutes to boil. Then, cool naturally in a well-ventilated area until cool to the touch (do not use ice to quickly cool the sections). Repeat the heating and boiling steps approximately 2-3 times. 3) Blocking Endogenous Peroxidases: Remove the sections from the slide box one by one and wipe off any remaining ddH2O around the tissue with absorbent paper (be careful not to allow the tissue to dry out). Add the 3% dH2O solution provided in the kit to the tissue and incubate in a humidified chamber protected from light for 15 minutes. After incubation, shake off any remaining liquid and wash the sections in PBS three times on a shaker, washing for 5 minutes each. 4) Blocking: Prepare a 1% BSA solution using BSA powder as the blocking solution. Remove the sections from the cassette and lay them flat in a humidified chamber. Apply the blocking solution dropwise to the tissue and incubate in a 37°C oven for 30 minutes. After blocking, remove the sections and rinse them three times with PBS for 5 minutes each. 5) Apply the primary antibody: Remove the sections and remove any excess liquid with absorbent paper. Place the sections in a humidified chamber and add the pre-prepared primary antibody solution of appropriate concentration. Incubate at 4°C overnight. 6) Equilibrate: Remove the humidified chamber from 4°C the next day and equilibrate at room temperature for 30 minutes. 7) Recover the primary antibody: Carefully recover the primary antibody and reuse it. 8) Add the secondary antibody: Dilute the secondary antibody in the 1% BSA prepared in step 4 and incubate in a 37°C oven for 1 hour. After incubation, carefully recover the secondary antibody and reuse it. Rinse the sections with PBS solution three times, 5 minutes each time; 9) DAB staining: prepare DAB colorimetric solution. Prepare DAB staining working solution according to the ratio of A solution: B solution = 1:20. After taking out the sections, add a suitable staining solution that can cover the entire tissue to the tissue, incubate in the dark for a suitable time. Then rinse off the excess staining solution with running water. Note: When preparing the DAB staining working solution, it is necessary to keep it away from light throughout the process, wrap the ep tube with tin foil, and it needs to be prepared immediately before use and cannot be reused. After incubation, rinse the sections with PBS solution three times, 5 minutes each time; 10) Nuclear staining: Add hematoxylin solution to the tissue sections, observe dynamically under the microscope to adjust the staining time, and keep the staining time consistent for each section. Then rinse the sections with PBS three times, 5 minutes each time; 11) Dehydration: Place the tissue sections in 70% ethanol solution for 5 seconds, 80% ethanol solution for 5 seconds, 90% ethanol solution for 5 seconds, 100% ethanol solution for 1 minute, and xylene solution for 3 minutes; 12) Sealing: After removing the sections from the xylene, wipe off the remaining xylene around the tissue, then quickly add neutral gum solution and cover with a coverslip to seal the sections.

[0035] 1.1.3 Tissue protein extraction 1) Weighing: After removing the myocardial tissue from the ultra-low temperature (-80°C) freezer, weigh 20 mg of tissue on a scale and quickly place the remaining tissue in a liquid nitrogen tank to prevent degradation. 2) Prepare tissue lysis buffer: Prepare lysis buffer according to the ratio of tissue lysis buffer: protease inhibitor: phosphatase inhibitor = 100 μL: 1 μL: 1 μL. For every 20 mg of tissue, add 400 μL of tissue lysis buffer. 3) Homogenize: Wash the grinding magnetic beads with ddH2O. After washing, place the clean magnetic beads in an EP tube containing the tissue and lysis buffer. Use a homogenizer to grind the tissue 6-10 times (make sure to balance the placement). 4) Centrifugation: After completion, use clean tweezers to remove the magnetic beads from each EP tube and centrifuge at 4°C, 12,000 rpm / min, for 10 min. 5) Aliquot: After completion, carefully remove the supernatant and place it in a -20°C freezer until needed.

[0036] 1.1.4 Prepare electrophoresis gel 1) Clean and dry the glass plates and electrophoresis combs needed for gel preparation. Align the long and short sides of the glass plates and secure them to the gel casting rack. 2) Select a precast gel with the appropriate concentration based on the molecular weight of the target protein. Prepare the lower gel using a ratio of 100:100:1 lower gel: lower gel buffer: coagulant. Each gel requires 8-9 mL of the lower gel mixture. After adding the lower layer of gel, add anhydrous ethanol sealing glue to flatten the liquid surface. 3) Top layer gel preparation: Combine different percentages of top layer gel solution, top layer gel buffer, and coagulant in a ratio of 100:100:1. Add to a beaker, mix thoroughly, and pour into the glass plate. Once filled, immediately insert the comb, taking care not to create bubbles. 4) Place the prepared gel rack in the electrophoresis tank and pour the electrophoresis fluid (prepare it as needed), taking care not to leak, then vertically remove the comb. 5) Sample loading: Use a 10μL pipette tip to aspirate the sample and marker and carefully load the sample into the appropriate lane. The sample volume should be approximately 5-10μL. 6) Electrophoresis: Fill the electrophoresis tank with freshly prepared electrophoresis buffer, close the lid, and set the parameters: voltage 160V, time 50 minutes. 7) Membrane Transfer: Place the glass plate after electrophoresis in ddH2O. After cutting the gel, place the gel on a NC membrane soaked in equilibration buffer. Clamp the two sides with sponges and transfer clamps. After clamping, place the membrane in a transfer apparatus and set the transfer time according to the molecular weight. 8) Blocking: Quickly place the transferred membrane in an incubation box containing 5% milk blocking buffer and incubate on a shaker for 1 hour (be careful not to dry the membrane during this process). 9) After the incubation, wash the membrane three times with TBST solution for 9-10 minutes each. Add the primary antibody to each incubation box and incubate overnight at 4°C. 10) The next morning, recover the primary antibody incubation solution and wash the membrane three times with TBST solution for 10 minutes each. Then, add rabbit / mouse secondary antibody incubation solution and incubate on a shaker for 1 hour; 11) Wash the membrane three times with TBST solution, each time for 9-10 minutes; 12) Band development: Prepare 2 mL of the luminescent solution at a 1:1 ratio, evenly coat the membrane, select appropriate imaging parameters, and capture images; 13) Data analysis: Calculate the expression levels of each target protein using ImageJ software.

[0037] 1.2 Experimental Results 1.2.1 OGTT and ITT The rats in the NC group and HINS group were fed a normal diet and a high-fat diet for 16 weeks, respectively. The fasting insulin and blood glucose levels of the rats were measured at 16 weeks, and the HOMA-IR values ​​were calculated. There was no significant difference in fasting blood glucose between the two groups (P>0.05). Figure 1 The fasting insulin level of rats in the HINS group was significantly increased (18.54 ± 4.51 μLU / mL vs. 10.27 ± 2.47 μLU / mL, P = 0.014), and the HOMA-IR index and body weight were significantly increased (P ≤ 0.05). Figure 1At 16 weeks, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed. The results of OGTT showed that there was no significant difference in fasting blood glucose levels between the two groups of rats. However, blood glucose levels in the HINS group were significantly increased at 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min (P ≤ 0.05). The blood glucose level dropped to normal levels more slowly, and the area under the curve in the HINS group was significantly increased (P ≤ 0.05). Figure 1 Middle E); The results of the ITT experiment showed that there was no significant difference in blood glucose between the two groups of rats at 0min and 15min. The fasting blood glucose levels of the HINS group were significantly higher than those of the NC group at 30min, 45min, 60min, 90min, 120min, and 150min (P≤0.05), and the area under the curve of the HINS group was significantly increased ( Figure 1 The above results indicate that the rat hyperinsulinemia model was successfully established.

[0038] 1.2.2 HE staining of heart tissue Studies have shown that hyperinsulinemia can damage the cardiac function of rats and mice, causing a certain degree of pathological changes. We used male rats fed a high-fat diet for 12 weeks as research subjects and used HE staining to detect the pathological changes in the heart tissue of rats fed a high-fat diet for 12 weeks and the control group of rats fed a normal diet. Figure 1 As shown, H&E staining was used to observe the structure of myocardial cells in the two groups. The myocardial cells in the NC group were spindle-shaped, regularly arranged, and stained evenly, without breakage or interstitial cell hyperemia and proliferation. The myocardial cells in the HINS group were arranged in a wavy pattern, with cellular and interstitial edema and vacuolation ( Figure 1 Middle G). This indicates that hyperinsulinemic rats fed a high-fat diet for 12 weeks exhibited certain pathological changes and could be used as an animal model for hyperinsulinemia-induced myocardial injury.

[0039] 1.2.3 Cardiac tissue immunohistochemistry and Western blotting results Western and RT-PCR results showed that the expression of AQP1 protein and mRNA in the heart tissue of the HINS group was higher than that of the NC group (p < 0.05). Immunohistochemistry results showed that the area of ​​AQP1-positive brown area in the heart of the HINS group was higher than that of the NC group ( Figure 1 (G, H).

[0040] Example 2 In vitro experimental verification of high concentration insulin-induced AQP1 protein expression in cardiac cells 2.1 Experimental Procedure 2.1.1 Cell culture (1) Cell culture conditions are shown in Table 1 Table 1: Cell culture conditions Cell growth requires a sterile environment.

[0041] (2) Cell recovery Prepare the complete culture medium required for the cells in advance. First, add 4 mL of complete culture medium to a 15 mL centrifuge tube. Then, remove the frozen cells from the -80°C freezer and quickly thaw them in water at approximately 37°C (rapid cell freezing solution can damage cells at room temperature). After thawing, pipette the cells into a 15 mL centrifuge tube containing complete culture medium on a sterile workbench. Centrifuge at 1000 rpm / min, level, and centrifuge for 5 minutes. Discard the supernatant, retain the cell pellet, and add 4 mL of complete culture medium. Use a sterile pipette to repeatedly pipette the cells to homogenize the suspension. Then, pipette the suspension into a culture flask, observe under a microscope, and return the tube to the incubator.

[0042] (3) Replace cell culture medium After disinfecting your hands with alcohol, remove the cells from the incubator and observe their growth under a microscope. When replacing the culture medium, remove the old medium with a pipette and rinse the flask with 2 mL of sterile PBS buffer or trypsin to remove dead cells. Discard the rinse solution and add 4 mL of complete culture medium to each flask. Return the flask to the incubator, following the aseptic procedure throughout.

[0043] (4) Cell passaging When the cells grow to an appropriate density (70%-80%), they need to be passaged to ensure that the cells have an appropriate growth rate. First, disinfect your hands, observe the cell growth density, remove the old culture medium with a pipette, add 2mL of trypsin to each culture flask to digest the cells, and observe the cell status dynamically under a microscope. When the cells lose their normal morphology and fall off from the flask wall in a round shape, add 2mL of complete culture medium (to avoid damaging the cells by digesting for too long). Then, use a pipette to blow the wall of the culture flask to blow the attached cells into the mixture of complete culture medium and trypsin, and then transfer them to a centrifuge tube, centrifuge at 1000rpm / min for 5 minutes, discard the supernatant, keep the cell pellet, add an appropriate amount of complete culture medium, blow it to make it evenly distributed in the suspension, and add it to the culture flask according to the passage plan (pay attention to sterile operation throughout the process).

[0044] (5) Cell cryopreservation First, disinfect your hands and prepare a cell freezing solution gradient on a clean bench. Mix 90% fetal bovine serum and 10% cell-specific DMSO in a 9:1 ratio. After removing the cells, centrifuge them according to the passage procedure. Discard the supernatant and add the appropriate amount of cell freezing solution. Aliquot into cell-specific cryovials and perform a gradient cooling. The next day, store in a liquid nitrogen tank.

[0045] (6) Cell plating and insulin intervention Cell digestion, centrifugation, and passage procedures were the same as above. Cells were seeded at an appropriate density in six-well plates. When cells reached 70-80% confluence, the old culture medium was discarded and the cells were starved for 48 hours with DMEM high-glucose medium. Subsequently, the cells were treated with various concentrations of insulin for 24 hours.

[0046] (7) Cell viability assay 1) Cell digestion and centrifugation are the same as before; 2) After centrifugation of the cell suspension, discard the supernatant, add complete culture medium, and pipette thoroughly to evenly distribute the suspension. Add 100 μL of the suspension to each well of a 96-well plate, and place the 96-well plate in a cell culture incubator for continued incubation; 3) The next day, after the cells have adhered, discard the old culture medium, add 100 μL of serum-free culture medium to each well, starve the cells for 48 h, add the intervention solution, and add only PBS to the blank control wells; 4) After the intervention time, add 10 μL of CCK8 solution to each well, and then return the wells to the incubator for continued incubation; 5) Set the microplate reader to read the OD value of 570 mm and measure the reading value of the 96-well plate at 30 min, 1 h, 2 h, and 2.5 h, respectively.

[0047] 2.1.2 Cell protein extraction 1) Prepare 2L of 1×PBS; 2) Prepare cell lysis buffer in the following ratios: RIPA: PMSF: phosphatase inhibitor 100μL: 1μL: 1μL, mix well and set aside; 3) Label the EP tube; 4) After removing the cells from the cell culture incubator, discard the old culture medium, rinse the cells three times with PBS solution, aspirate the PBS, add 400μL of lysis buffer to each well of the six-well plate, thoroughly triturate with a 1mL pipette tip, and aspirate the liquid into the EP tube; 5) Centrifuge at 4℃, 3000rpm / min, for 5 minutes, transfer the resulting supernatant to a labeled EP tube, record the supernatant volume, and store at -80℃.

[0048] 2.1.3 Cell immunofluorescence 1) First cell slide; 2) Discard the culture medium, rinse with PBS for 3 times, and absorb the remaining liquid, and fix the cells with a special cell fixing solution for 5-10 min; 3) Discard the fixing solution, rinse with PBS for 3 times, 5 min each time, and absorb the PBS; 4) Add an appropriate amount of 1% Triton to the cell perforation for 15 min, and then rinse the cells with PBS for 3 min each time, 5 min each time; 5) Use 1% BSA to block the cells for 60 min; 6) Dilute the primary antibody according to the instructions, add 250 μL of the diluted primary antibody to each well, and incubate at 4°C overnight; 7) The next day, equilibrate at 37°C for 30 min, recover the primary antibody, which can be reused, and rinse the cells with PBS for 3 times, 5 min each time; 8) Add an appropriate amount of fluorescent secondary antibody, incubate at 37°C for 30 min, and then rinse with PBS for 3 times, 5 min each time; 9) Use DAPI containing an anti-quenching agent to mount the slide and stain the cell nucleus, and avoid light for 10 min; 10) Take pictures under a microscope and save them.

[0049] 2.2 Experimental results 2.2.1 CCK8 detection of cell viability after intervention with different concentrations of insulin Select 0 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, and 2500 ng / mL of insulin to intervene in cells for 24 h. The results are shown in the figure, and the insulin concentration from 0 ng / mL to 500 ng / mL basically does not affect the cell activity. When the insulin concentration is 1000 ng / mL, the cell activity is reduced to about 80%, and when the insulin concentration is 2500 ng / mL, it is only about 70%. Therefore, 0 ng / mL-500 ng / mL of insulin concentration gradient is used for subsequent experiments. Figure 2 C).

[0050] 2.2.2 Effect of different concentrations of insulin on key proteins for water and sodium-calcium ion regulation in H9c2 cells Use 0 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 250 ng / mL, and 500 ng / mL of insulin to intervene in H9c2 myocardial cells, and the results are shown in the figure. P-AKT, AQP1, and NHE1 all have the highest expression when the insulin concentration is 500 ng / mL, and the protein expression of P-AKT, AQP1, and NHE1 gradually increases as the insulin concentration increases from 0 ng / mL to 500 mg / mL. Therefore, 500 ng / mL is selected as the subsequent cell intervention concentration. Figure 2 D, E).

[0051] 2.2.3 High-concentration insulin intervention increases AQP1 expression in H9c2 myocardial cells Western blotting showed that compared with the control group, the expression of AQP1 in the myocardial cells of the insμLin intervention group was significantly increased ( Figure 2 (A, B in Chinese).

[0052] Example 3 Effects of AQP1 on Cardiomyocytes in a High Insulin Concentration Environment 3.1 Experimental Procedure 3.1.1 Confocal microscopy stack scanning to detect cell edema 1) Turn on the instrument power and light source: First, turn on the microscope and laser. Next, open the computer and software, set the wavelength of the fluorescent dye excitation light, and select the appropriate filter to obtain sufficient fluorescence signal. 2) Set the corresponding scanning mode: Adjust the objective lens magnification to locate the cells to be examined under the fluorescence microscope. Then switch to scanning mode and adjust the focus to obtain a clear confocal image. 3) Observe the image under the microscope: ① Select "Z-Stack"; ② Adjust the thickness and number of layers for each image scan; ③ Press the "Start" button to obtain a complete image under the microscope. 4) Shutdown: After the instrument has completed the sample, first turn off the laser so that the computer can continue to process the image and data. 5) Use ZEN software for image processing and analysis.

[0053] 3.1.2 Detection of sodium ion concentration by cell atomic absorption spectrometry 1) Cell suspension collection: Remove cells from the incubator, discard the old culture medium, and rinse 2-3 times with sterile PBS. Add 2 mL of EDTA to each well of a 6-well plate. Incubate in a 37°C incubator for 3 minutes, then remove and add 2 mL of complete culture medium to terminate digestion. Collect the EDTA-culture medium mixture into a 4 mL EP tube, label each well, and centrifuge at 1000 rpm for 5 minutes to collect the cell pellet. Add approximately 200 μL of sterile PBS to each tube (the appropriate amount of PBS depends on the number of cells). Disrupt the cells using an ultrasonic probe three times for 2 minutes each (the ultrasonication frequency is set to 20 Hz). After the cell suspension is broken by centrifugation, the supernatant is transferred to a new 2 mL EP tube. 2) Standard solution preparation: Dilute the standard solution into five concentrations of 8, 4, 2, 1, and 0.5 mM for later use. 3) Sample loading: Add 100 μL of the standard solution and prepared sample to a 96-well plate, respectively. Based on the absorbance of different wells, draw a standard curve and calculate the final concentration of each sample.

[0054] 3.1.3 Fluo-4AM calcium ion fluorescent probe detection 1) Take an appropriate amount of Fluo-4 AM stock solution and dilute it with PBS to a 2 WM working solution. Note: The working solution must be prepared immediately before use and should not be repeatedly frozen and thawed. 2) Remove the culture medium from the adherent cells and rinse three times with PBS. Note: The serum in the culture medium contains esterases that can break down Fluo-4 AM into Fluo-4, and phenol red can increase background fluorescence. 3) Add the Fluo-4 AM working solution, using a volume sufficient to fully cover the cells. A typical volume for a six-well plate is 1 mL. 4) Incubate at 37°C for 30 minutes to load the fluorescent probe. Note: If this is your first experiment, try observing the fluorescence effect for 30 minutes. If there is a lot of cell death, shorten the time or lower the incubation temperature appropriately. If the fluorescence is too weak, extend the incubation time appropriately. 5) Then wash three times with PBS. After washing, consider incubating for an additional 20-30 minutes to ensure that Fluo-4AM is completely converted to Fluo-4 in the cells. 6) Use a laser confocal microscope to detect the green-specific fluorescence intensity of Fluo-4 in the cells to determine changes in intracellular calcium ion concentration.

[0055] 3.1.4 RHOD-2AM mitochondrial fluorescent probe detection 1) Take an appropriate amount of Rhod-2AM stock solution and dilute it with PBS to a 1μM working solution. Note: The working solution must be prepared immediately before use and should not be repeatedly frozen and thawed. 2) Remove the culture medium from the adherent cells and rinse three times with PBS. 3) Add the Rhod-2AM working solution, using a volume sufficient to fully cover the cells. A six-well plate typically requires 1mL of solution. 4) Incubate at 37°C for 30 minutes to allow the fluorescent probe to be loaded. 5) Wash three times with PBS. 6) Measure the red-specific fluorescence intensity of Rhod-2AM in the cells using a laser confocal microscope to determine the intramitochondrial calcium concentration.

[0056] 3.1.5 Detection of mitochondrial membrane potential in JC-1 cells 1) First, prepare the positive control working solution, dilute it at a ratio of 1:1000, add it to the cells, and place it in the incubator for about 30 minutes; 2) Then prepare the JC-1 staining working solution, add 50μL of JC-1 staining working solution (200X) to 8mL of ddH2O, mix well, and then add 2mL of JC-1 staining buffer (5X) to mix evenly; 3) After the positive control working solution is incubated in the cell culture incubator, rinse the cells three times with PBS, each for 5 minutes; 4) Add 500μL of complete cell culture medium to each well; 5) Then, add 500μL of JC-1 staining working solution to each well and return to the 37℃ incubator to incubate for 30 minutes; 6) JC-1 staining buffer working solution is diluted according to the ratio of 1mL staining buffer (5×): 4mL Prepare the JC-1 staining solution in a ratio of 50:1 to 100:1 (prepared with ddH2O) (must be prepared fresh and used immediately); 7) After incubation at 37°C, discard the supernatant and wash twice with the prepared JC-1 staining buffer; 8) Add 2 mL of cell culture medium to each well; 9) Capture and save images using a fluorescence microscope.

[0057] 3.2 Experimental Results 3.2.1 High-concentration insulin treatment of H9c2 cardiomyocytes resulted in cellular edema Calcein AM (calcein acetoxymethyl ester) is a green fluorescent probe with strong hydrophobicity. It penetrates the cell membrane and enters the living cell, emitting strong green fluorescence, which specifically fluorescently labels the living cell. The confocal microscopy stack scanning results showed that the number of stack scanning layers of cells in the high-concentration insulin intervention group increased, and the cross-sectional area of ​​the brightest layer of cells also increased, indicating that the cell volume increased ( Figure 3 Middle A).

[0058] 3.2.2 Abnormal expression of sodium and calcium regulatory proteins after high-concentration insulin treatment in H9c2 cardiomyocytes Western results showed that compared with the control group, the levels of NHE1 and SGLT1 proteins in the high-concentration insulin intervention group were significantly increased, while the levels of NKA, NCX, and SERCA proteins were decreased ( Figure 3 Middle B).

[0059] 3.2.3 Sodium and calcium overload after high-concentration insulin treatment of H9c2 cardiomyocytes Fluo-4 AM (Fluo-4 Calcium) is a specific calcium ion fluorescent probe. Fluo-4 AM itself has almost no fluorescence. Once it enters the cell and is hydrolyzed by intracellular esterase, it produces Fluo-4 which can emit green fluorescence after binding with calcium ions. Confocal microscope results show that compared with the control group, the Fluo-4 AM signal in the insulin intervention group increases. Figure 3 C、 Figure 4 A).

[0060] 3.2.4 Insufficient calcium content in mitochondria of H9c2 myocardial cells after high-concentration insulin intervention Rhod-2 AM is a specific mitochondrial fluorescent probe that can effectively increase the mitochondrial calcium fluorescence signal by 80-100 times, and its signal intensity is the strongest among all mitochondrial calcium ion probes. Therefore, Rhod-2 AM is usually used as a specific probe to detect the calcium ion content in mitochondria. Confocal microscope results show that compared with the control group, the Rhod-2 AM signal in the mitochondria of the high-concentration insulin intervention group is weakened, while the expression of ROS in the cells increases. Figure 4 B).

[0061] 3.2.5 High-concentration insulin causes mitochondrial function damage in H9c2 myocardial cells Western results show that compared with the control group, the expression of PGC1a and OPAl proteins in the insulin intervention group is significantly reduced, while the level of p-DRP1 / DRP1 protein is significantly increased.

[0062] 3.2.6 High-concentration insulin causes mitochondrial membrane potential damage in H9c2 myocardial cells JC-1 is a fluorescent probe for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix, forming polymers and producing red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix, and at this time, JC-1 is in monomer form, producing green fluorescence. The transition of JC-1 from red fluorescence to green fluorescence is usually used as an indicator for detecting the decline of cell membrane potential. Compared with the control group, the green fluorescence in the high-concentration insulin intervention group is enhanced, the red fluorescence is reduced, and the mitochondrial membrane potential is decreased. Figure 4 C).

[0063] Example 4 siRNA-AQP1 improves high-concentration insulin-induced myocardial cell damage 4.1 Experimental process 4.1.1 Transfection of si-AQP1 in H9c2 cells 1) Seed H9c2 cells in a 6-well plate at 5 × 10 cells per well and transfect the next day; 2) Prepare serum-containing culture medium without the double-antibody; 3) Aspirate the old culture medium from the 6-well plate and rinse the cells twice with PBS; 4) Add 245 μL of Opti-T and 5 μL of Lipofectamine 3000 to each well of the 6-well plate, pipette well, incubate at room temperature for 5 minutes, and set aside; 5) Dilute the AQP1 inhibitor negative control and AQP1 inhibitor to the working concentration in 250 μL of DEPC water according to the manufacturer's instructions. Similarly, add 245 μL of Opti and 5 μL of AQP1 inhibitor to each well of a 6-well plate. Mix thoroughly by pipetting, incubate at room temperature for 5 minutes, and set aside. 6) Mix the diluted Lipo 3000-Opti mixture and the diluted inhibitor-Opti mixture and incubate at room temperature for 20 minutes to prepare the AQP1 inhibitor-Lipo 3000 complex. 7) Discard the old culture medium in the 6-well plate, rinse the cells twice with PBS, and add 1.5 mL of Opti solution to each well, followed by 500 μL of the AQP1 inhibitor-Lipo 3000 complex, dripping in a circular pattern around the edges. Add 2 mL of Opti solution to the blank control well. 8) Return the 6-well plate to the cell culture incubator and observe the cells dynamically. After incubation for 4-6 hours, aspirate the old culture medium and replace with 2 mL of culture medium without the antibody and incubate for 48 hours. 9) After the intervention period, extract cell protein from each well.

[0064] 4.2 Experimental Results 4.2.1 Verification of si-AQP1 knockdown sequence Western blot results showed that 48 hours after si-AQP1 transfection, AQP1 protein expression in the AQP1-siRNA-2 sequence decreased most significantly, decreasing by approximately 73% compared to the ctlsiRNA group, a statistically significant difference (P < 0.05). Cy5 is a red fluorescent marker that can specifically fluorescently label knockdown sequences. Cells stained with a cys fluorescent probe and photographed using a confocal microscope showed that the specific red fluorescence of Cy5 could successfully fuse with the green fluorescence that stains the cytoplasm. These results indicate that the intracellular si-AQP1 knockdown sequence was successfully constructed ( Figure 5 (A, B in Chinese).

[0065] The si-AQP1 target sequence is: GCAGCAGAGTAAAGGTCAT.

[0066] 4.2.2 si-AQP1 reduces cellular edema under insulin intervention Next, we examined the edema of cells after si-AQP1 treatment under ins intervention. Confocal microscopy results showed that compared with the ins intervention group, the number of cell scan layers in the ins+si-AQP1 group decreased from 19 to 13, and the cross-sectional area of ​​the brightest layer of the scanned cells was also significantly reduced ( Figure 5 Middle C).

[0067] 4.2.3 si-AQP1 can alleviate cellular water, sodium, and calcium ion imbalances under insulin intervention Then, the key regulatory proteins of water, sodium and calcium balance were detected by western blotting. The results showed that compared with the ins pre-treated group, the expression levels of NHE1 and SGLT1 proteins, which transport sodium ions into the cells, were significantly decreased in the ins+si-AQP1 group (P < 0.05), while the expression levels of NKA, SERCA and NCX proteins, which excrete sodium and calcium ions out of the cells, were increased (P < 0.05). The expression levels of PGC1α, the key protein of mitochondrial biogenesis, MFN1 and OPA1 were significantly increased, while the expression of DRP1, the mitochondrial fission protein, was decreased (P < 0.05). Figure 6 Middle A).

[0068] 4.2.4 si-AQP1 alleviates cellular sodium and calcium overload and mitochondrial calcium deficiency under insulin intervention The sodium and calcium ion concentrations in each group of cells were further observed. Confocal microscopy results showed that compared with the insulin intervention group, the green fluorescence signal FLUO-4AM of calcium ions in the myocardial cytoplasm of the ins+si-AQP1 group decreased, and the sodium ion concentration in the cells detected by atomic absorption spectrometry decreased (P < 0.05); the red fluorescence signal RHOD-2AM of calcium ions in the mitochondria increased ( Figure 6 Middle B).

[0069] 4.2.5 si-AQP1 alleviates the decrease in mitochondrial membrane potential in myocardial cells under insulin intervention The results of JC-1 mitochondrial membrane potential staining showed that compared with the ins intervention group, the red fluorescence in the ins+si-AQP1 group was enhanced, while the green fluorescence was decreased, and the mitochondrial membrane potential was increased ( Figure 7 ).

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. Application of AQP1 protein as a target in the preparation of drugs for the treatment of hyperinsulinemia-induced myocardial injury.

2. The use according to claim 1, characterized in that The drug exerts its effects by inhibiting the expression level of AQP1 or the activity of AQP1.

3. The use according to claim 2, characterized in that The inhibitor for inhibiting the expression of AQP1 or the activity of AQP1 is Tc-AQP1 or siRNA.

4. The use according to claim 3, characterized in that The inhibitor is siRNA.

5. The use according to claim 4, characterized in that The target sequence of the siRNA is: GCAGCAGAGTAAAGGTCAT.

6. The use according to claim 1, characterized in that The hyperinsulinemia myocardial injury includes any one of myocardial cell edema, sodium and calcium ion overload or mitochondrial dysfunction.

7. A pharmaceutical composition for treating hyperinsulinemia myocardial injury, characterized in that: The invention comprises an AQP1 inhibitor and a pharmaceutically acceptable carrier.

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