Use of AQP1 in preparation of drugs for treating hyperinsulinemic myocardial injury

Abstract

The application provides an application of AQP1 in preparation of a drug for treating hyperinsulinemic myocardial injury, relates to the field of biological medicines, and uses an inhibitor related to the AQP1 protein as a target to inhibit the AQP1 protein, thereby effectively reducing myocardial edema, improving ion homeostasis imbalance and repairing mitochondrial dysfunction, so that the effect of treating hyperinsulinemic myocardial injury is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of AQP1 in the preparation of drugs for treating myocardial injury in patients with hyperinsulinemia. Background Technology

[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 type of cardiomyopathy independent of other cardiovascular diseases such as hypertension, coronary artery disease, valvular heart disease, and congenital heart disease. Clinically, it is characterized by early diastolic dysfunction, late systolic dysfunction, and heart failure. Many metabolic environments associated with diabetes promote myocardial damage and cardiomyocyte death. Currently, it is believed that the main factors triggering DCM in T2DM are insulin resistance and hyperinsulinemia. Hyperinsulinemia can mediate myocardial fiber degeneration and left ventricular remodeling, leading to increased ventricular wall stiffness, decreased compliance, and inducing early diastolic myocardial dysfunction. Numerous studies have shown that myocardial insulin resistance and hyperinsulinemia are closely related to sodium and calcium ion homeostasis in cells. Ion homeostasis in cardiomyocytes is regulated by ion pumps on the cell membrane surface, and sodium and calcium ions are key factors involved in regulating ion pumps and myocardial function. Abnormal transduction of the insulin signaling pathway in cardiomyocytes disrupts sodium and calcium ion homeostasis by affecting the expression of ion pumps. When sodium and calcium ions are overloaded in cells and mitochondrial calcium levels are reduced, mitochondrial dysfunction and increased oxidative stress occur, ultimately leading to cell death. Sodium ions and water molecules maintain a dynamic balance in the human body. When sodium ion levels rise in cells, water molecules enter the cell via free diffusion and channel transport, causing cellular edema. Fluid balance plays a crucial role in maintaining cardiomyocyte function. Edema affects myocardial contractility and relaxation by increasing ventricular wall stiffness and decreasing ventricular wall compliance; furthermore, edema can affect ventricular electrical signal conduction by influencing the ionic composition of cardiomyocytes, thus leading to arrhythmias. Therefore, myocardial fluid balance is essential for maintaining normal cardiac function.

[0003] AQPs (aquaporins) are a class of important molecular channel proteins that maintain water balance in the myocardium. In 1968, Benga et al. discovered these small proteins involved in water transport and subsequently named them aquaporin channel proteins (AQPs). Their expression significantly increases cell membrane permeability. AQPs are widely distributed throughout the body's organs, mediating the transport of small molecules such as water, O2, H2O, and N2. Currently, 13 AQP isoforms with homologous molecular structures have been identified in mammals. They are approximately 320 amino acid residues long, with a molecular weight of about 28 kDa, and exist as tetramers on the cell membrane. AQP expression varies across different species and organs, with different isoforms coordinating their expression to maintain homeostasis. The main isoforms expressed in the myocardium include AQP1, AQP4, and AQP7. AQPs play a crucial role in maintaining myocardial water balance. Water molecules can flow between cells via free diffusion, ion channel transporters, or aquaporins. Under physiological conditions, one-third of the water flowing into the myocardial tissue via capillaries is regulated by aqueous quicklime (AQPs). In 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, by mediating mitochondrial swelling and increased oxygen diffusion distance, affects the normal oxidative phosphorylation and ATP synthesis capacity of cardiomyocytes, leading to cardiac dysfunction. AQP1 is an important subtype for maintaining myocardial water balance. Current research has found that AQP1 is closely related to many cardiovascular diseases such as myocardial infarction, myocardial ischemia-reperfusion injury, myocardial ischemia-hypoxia, and cardiopulmonary bypass surgery; increased AQP1 expression mediates the occurrence and development of myocardial edema.

[0004] Currently, the mechanism of action and application of AQP1 in myocardial injury caused by hyperinsulinemia are unclear.

[0005] Therefore, this invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of AQP1 in the preparation of drugs for treating myocardial injury in hyperinsulinemia. AQP1 protein is used as a target, and related inhibitors are employed to inhibit it, thereby effectively reducing myocardial edema, improving ion homeostasis imbalance, and repairing mitochondrial dysfunction, thus exerting a therapeutic effect on myocardial injury in hyperinsulinemia.

[0007] To achieve the objectives of this invention, the following solution is provided:

[0008] Application of AQP1 protein as a target in the preparation of drugs for treating myocardial injury in patients with hyperinsulinemia.

[0009] Furthermore, the drug exerts its effect by inhibiting the expression level or activity of AQP1.

[0010] Furthermore, the inhibitor that suppresses the expression or activity of AQP1 is either Tc-AQP1 or siRNA.

[0011] Furthermore, the inhibitor is siRNA.

[0012] Furthermore, the target sequence of the siRNA is: GCAGCAGAGTAAAGGTCAT.

[0013] Furthermore, the hyperinsulinemia-related myocardial injury includes myocardial cell edema, sodium-calcium ion overload, or mitochondrial dysfunction.

[0014] The present invention also provides a pharmaceutical composition for treating myocardial injury in patients with hyperinsulinemia, comprising an AQP1 inhibitor and a pharmaceutically acceptable carrier.

[0015] The present invention also provides an in vitro diagnostic kit for detecting myocardial injury in hyperinsulinemia, the kit comprising: a reagent for detecting AQP1 expression levels.

[0016] Furthermore, the reagents include specific antibodies or nucleic acid probes against AQP1.

[0017] The embodiments of the present invention have the following technical effects:

[0018] This invention targets the AQP1 protein and further uses siRNA to inhibit AQP1 expression or activity, which can improve high-insulin-induced cardiomyocyte edema, cellular ion homeostasis imbalance, and mitochondrial dysfunction. Drugs that inhibit AQP1 expression can be used to treat myocardial injury in hyperinsulinemia. This not only provides a new drug target for the prevention or treatment of myocardial injury in hyperinsulinemia, but also provides a new approach for the treatment, prevention, and related drug preparation of this disease. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1Heart tissues of animals with hyperinsulinemia model; 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 body 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 immunohistochemical 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.

[0021] Figure 2 The results of high-concentration insulin inducing AQP1 protein expression in cardiomyocytes; Figure A shows the trend of AQP1 protein changes in cardiomyocytes in two groups; Figure B shows the immunofluorescence staining results of AQP1 in two groups; Figure C shows the cck8 results after different concentrations of insulin gradient intervention in cardiomyocytes; Figures D and E show the trends of NHE1, AKT, p-AKT, and AQP1 protein changes after different concentrations of insulin intervention.

[0022] Figure 3 The results show the effects of AQP1 on water, sodium-calcium balance and mitochondrial function in cardiomyocytes under high insulin concentration. Figure A shows cell edema after high insulin intervention in cardiomyocytes, Figure B shows water and sodium ion metabolism disorder after high insulin intervention in cardiomyocytes, and Figure C shows sodium and calcium ion expression after high insulin intervention in cardiomyocytes.

[0023] Figure 4 The results show the effects of AQP1 on water, sodium-calcium balance and mitochondrial function in cardiomyocytes under high insulin concentration. Figure A shows the calcium ion and ROS content in mitochondria after high insulin intervention in cardiomyocytes. Figure B shows the changes in key indicators of mitochondrial function after high insulin intervention in cardiomyocytes. Figure C shows the changes in mitochondrial membrane potential of JC-1 cells after high insulin intervention in cardiomyocytes.

[0024] Figure 5 si-AQP1 can alleviate the water, sodium-calcium imbalance and mitochondrial dysfunction induced by high-concentration insulin 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 intervention with high-concentration insulin.

[0025] Figure 6 : si-AQP1 can alleviate the water, sodium-calcium imbalance and mitochondrial dysfunction induced by high concentration insulin in cardiomyocytes; Figure A shows the sodium and calcium ion content of si-AQP1 cells and mitochondria under high concentration insulin intervention; Figure B shows the effect of si-AQP1 on the reduction of changes in mitochondrial membrane potential in cardiomyocytes under high concentration insulin intervention.

[0026] Figure 7 The effect of si-AQP1 on reducing cellular sodium and calcium overload and mitochondrial calcium content under insulin intervention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0028] In a first aspect, the present invention provides the application of using AQP1 protein as a target in the preparation of drugs for treating myocardial injury in patients with hyperinsulinemia.

[0029] In some embodiments, the drug exerts its effect by inhibiting the expression level or activity of AQP1.

[0030] In some embodiments, the inhibitor that suppresses AQP1 expression or AQP1 activity is Tc-AQP1 or siRNA.

[0031] In some embodiments, the inhibitor is siRNA.

[0032] In some embodiments, the target sequence of the siRNA is: GCAGCAGAGTAAAGGTCAT.

[0033] In some embodiments, the hyperinsulinemia-related myocardial injury includes any one of cardiomyocyte hypertrophy, sodium-calcium ion overload, or mitochondrial dysfunction.

[0034] On the other hand, the present invention also provides a pharmaceutical composition for treating myocardial injury in patients with hyperinsulinemia, comprising an AQP1 inhibitor and a pharmaceutically acceptable carrier.

[0035] On the other hand, the present invention also provides an in vitro diagnostic kit for detecting myocardial injury in hyperinsulinemia, the kit comprising: a reagent for detecting AQP1 expression levels.

[0036] In some embodiments, the reagent includes a specific antibody or nucleic acid probe against AQP1.

[0037] The following is a detailed explanation using specific embodiments:

[0038] Example 1: Determination of AQP1 protein expression level in cardiac tissue of a model animal with hyperinsulinemia

[0039] 1.1 Experimental Procedure

[0040] 1.1.1 Animal grouping and tissue collection

[0041] 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). Rats in the NC group were fed a normal diet, while rats in the HINS group were fed a 60% kcal high-fat diet. After 16 weeks of feeding, the body weight of rats in each group was measured weekly. Every 4 weeks, the rats were placed in metabolic cages for 24 hours to monitor their metabolic status. Blood was collected from the inner canthus of the rats' eyes, centrifuged, and serum was used to detect changes in lipid profiles, liver and kidney function, and insulin levels.

[0042] Criteria for establishing a hyperinsulinemia model: elevated fasting plasma insulin levels in rats with a statistically significant difference between the two groups. Subsequently, OGTT and ITT tests were performed, using the following methods:

[0043] (1) Oral glucose tolerance test (OGTT)

[0044] An oral glucose tolerance test (OGTT) was performed on rats before sampling to assess their glucose metabolism. Rats were weighed the day before and fasted overnight, but allowed free access to water. The following morning, fasting blood glucose levels at the tail tip of each group were measured and recorded as the 0-minute value. Then, rats were sequentially administered a prepared 30% glucose solution (2 g / kg) via gavage, with the gavage time recorded for each rat. Subsequently, blood glucose levels were measured at 30, 60, 90, 120, 150, and 180 minutes for each group. Curves were plotted, and the area under the curve was calculated for comparison between groups.

[0045] (2) Insulin Tolerance Test (ITT)

[0046] Before sampling, an ITT (intracytoplasmic transluminal test) was performed to assess pancreatic function in rats. The rats were weighed the day before, and the insulin dosage for each group was calculated. Rats in each group were fasted for 6 hours but allowed free access to water. A 30% glucose solution was prepared to prevent hypoglycemia. At dawn the following day, fasting tailbone blood glucose levels were measured and recorded as the 0-minute value. Based on body weight, rats were intraperitoneally injected with NovoRapid 30 insulin at a dose of 0.5 U / kg. Blood glucose levels were measured at 15, 30, 45, 60, 90, 120, and 150 minutes after insulin injection. Curves were plotted, and the area under the curve (AUC) was calculated to compare differences between the two groups.

[0047] After successfully establishing the hyperinsulinemia model, rats were fasted overnight and anesthetized with an intraperitoneal injection of 500 mg / kg tribromoethanol. The beating heart tissue was then removed and placed in sterile saline to allow residual blood to drain from the heart tissue using its remaining pulsation. The tissue was then weighed and the values ​​recorded. Tissue from the apex of the heart was removed with a scalpel and fixed in 4% paraformaldehyde. The remaining tissue was placed in EP tubes and stored at -80°C.

[0048] 1.1.2 H&E staining and immunohistochemical staining

[0049] (1) H&E staining

[0050] 1) Remove the wax block from the embedding machine at -20℃, trim it until the cut surface is smooth, and then section it, being careful not to curl the sections; the thickness should be 4-5μm. 2) Place the sectioned tissue slides on a glass slide and immerse them in 42℃ warm water to fully expand the tissue sections. 3) Immerse the expanded tissue sections in xylene twice for 10 minutes each time to dewax them. 4) Immerse the xylene-soaked tissue sections in anhydrous ethanol for 5 minutes to dehydrate them, then immerse them in 90%, 80%, and 70% ethanol for 5 minutes each time, and then immerse them in ddH2O. 5) immerse the hydrated tissue sections... 6) Immerse the sections in hematoxylin staining solution for 3-5 minutes; 7) Rinse off the hematoxylin with tap water for about 2 minutes, then place the sections in 1% hydrochloric acid ethanol for 2 seconds to differentiate, rinse the sections with tap water for 1 minute, and soak in ddH2O for 5 minutes to invert blue; 8) Place the tissue sections in eosin staining solution for 1 minute; 9) Rinse the sections with ddH2O to remove excess eosin staining solution, then perform gradient dehydration of the sections: 80% ethanol for 2 seconds - 90% ethanol for 2 seconds - anhydrous ethanol for 10 seconds - xylene for 3 minutes; 10) Immediately after removing from xylene, add neutral resin, cover with a coverslip and seal.

[0051] (2) Immunohistochemical staining

[0052] 1) Dewaxing: First, bake the tissue sections at 60℃ for 2 hours, then soak them in xylene I solution for 10 min, xylene II solution for 10 min, anhydrous ethanol for 5 min, 90% ethanol solution for 5 min, 80% ethanol solution for 5 min, 70% ethanol solution for 5 min, and ddH2O for 5 min. 2) Antigen retrieval: Prepare a 1x Tris-EDTA solution, place the sections in the solution, and microwave on high for 5 min until boiling. Then allow them to cool naturally in a ventilated area until they are no longer hot to the touch (do not use ice to cool them quickly). Repeat the heating and boiling steps approximately 2-3 times. 3) Blocking endogenous peroxidase: Remove the sections one by one from the cassette, and wipe away any residual ddH2O around the tissue with absorbent paper (be careful not to let the tissue dry out). Add 3% H2O solution from the kit to the tissue and incubate in a humidified chamber in the dark for 15 min. 4) Blocking: Prepare a 1% BSA solution using BSA powder as the blocking solution. Remove the slides from the box, place them flat in a humidified chamber, add the blocking solution, and incubate at 37°C for 30 minutes. After blocking, remove the slides and rinse them three times with PBS solution for 5 minutes each time. 5) Adding primary antibody: Remove the slides and absorb excess liquid with absorbent paper. Place them in a humidified chamber and add a pre-prepared primary antibody solution of appropriate concentration. Incubate overnight at 4°C. 6) Equilibration and rewarming: The next day, remove the humidified chamber from 4°C and equilibrate at room temperature for 30 minutes. 7) Recovering primary antibody: Carefully recover the primary antibody, which can be reused. 8) Adding secondary antibody: Dilute the secondary antibody with the 1% BSA prepared in step 4, incubate at 37°C for 1 hour, and carefully recover the secondary antibody, which can be reused. 9) DAB staining: Prepare DAB staining solution. Prepare DAB staining working solution according to the ratio of solution A: solution B = 1:20. After removing the section, add an appropriate amount of staining solution to cover the entire tissue and incubate in the dark for an appropriate time. Then rinse off the excess staining solution with running water. Note: The DAB staining working solution must be prepared in the dark throughout the process. Wrap the EP tube with aluminum foil and prepare it fresh for each use. After incubation, rinse the section three times with PBS solution for 5 minutes each time. 10) Nuclear staining: Add hematoxylin solution to the tissue section and dynamically observe and adjust the staining time under a microscope to keep the staining time consistent for each section. Then rinse the sections three times with PBS, 5 min each time; 11) Dehydration: Place the tissue sections in 70% ethanol solution for 5 s, 80% ethanol solution for 5 s, 90% ethanol solution for 5 s, 100% ethanol solution for 1 min, and xylene solution for 3 min in sequence; 12) Mounting: After removing the sections from the xylene, wipe away the residual xylene around the tissue, then quickly add a neutral resin solution, cover with a coverslip and mount.

[0053] 1.1.3 Tissue protein extraction

[0054] 1) Weighing: After removing the myocardial tissue stored at -80℃, weigh 20mg of tissue on a balance. Quickly place the remaining tissue into a liquid nitrogen tank to prevent degradation. 2) Preparing tissue lysis buffer: Prepare the lysis buffer according to the ratio of tissue lysis buffer: protease inhibitor: phosphatase inhibitor = 100μL: 1μL: 1μL. 400μL of tissue lysis buffer is needed for every 20mg of tissue. 3) Homogenizing tissue: Clean the grinding magnetic beads with ddH2O. After cleaning, place the clean magnetic beads in an EP tube containing tissue and lysis buffer. Homogenize the tissue 6-10 times using a homogenizer (make sure to place them evenly). 4) Centrifugation: After centrifugation, remove the magnetic beads from each EP tube with clean tweezers. Centrifuge at 12000rpm / min for 10min at 4℃. 5) Aliquoting: After centrifugation, carefully collect the supernatant and store it in a -20℃ freezer for later use.

[0055] 1.1.4 Electrophoresis Gel

[0056] 1) Clean and dry the glass plates and electrophoresis combs needed for gel preparation. Align the long and short plates of the glass plates and fix them on the gel casting rack; 2) Select the appropriate percentage concentration of precast gel according to the molecular weight of the target protein. Prepare the lower gel according to the ratio of lower gel: lower gel buffer: coagulant = 100:100:1, requiring 8-9 mL of lower gel mixture per gel. 3) Top layer preparation: Prepare top layer solutions, top layer buffer, and coagulant in a ratio of 100:100:1, add to a beaker, mix well, and pour into a glass plate. Immediately insert the comb after filling, being careful not to generate air bubbles; 4) Place the prepared gel in the electrophoresis tank, pour in the electrophoresis buffer (prepare fresh for use), being careful not to leak, and then vertically pull out the electrophoresis comb; 5) Sample loading: Use a 10μL pipette tip to draw up the sample and marker, and carefully load the sample into the appropriate lane. The sample loading volume is approximately 5-10μL; 6) Electrophoresis: Fill the electrophoresis tank with freshly prepared electrophoresis buffer, cover it, and set the parameters: voltage 160V, time 50min; 7) Transfer: Place the electrophoretically processed glass plate in ddH2O, cut the gel, place the gel in an NC membrane soaked in equilibration buffer, clamp both sides with sponges, clamp with transfer clamps, and place in a transfer apparatus. Set the appropriate transfer time according to the molecular weight; 8) Blocking: Quickly place the transferred membrane into an incubation box containing 5% milk blocking buffer and shake for 1 hour (note that the membrane should not dry out during the entire process); 9) After incubation, wash the membrane three times with TBST solution, 9-10min each time. Add 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, 10min each time. 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, 9-10 minutes each time; 12) Band development: Prepare 2 mL of luminescent solution at a 1:1 ratio, apply evenly to the membrane, select appropriate parameters on the imager, and take pictures; 13) Data analysis: Calculate the expression level of each target protein using ImageJ software.

[0057] 1.2 Experimental Results

[0058] 1.2.1 OGTT Experiment and ITT Experiment

[0059] Rats in the NC group and HINS group were fed a normal diet and a high-fat diet, respectively, for 16 weeks. Fasting insulin and blood glucose levels were measured at 16 weeks, and HOMA-IR values ​​were calculated. There was no significant difference in fasting blood glucose between the two groups (P > 0.05). Figure 1In the HINS group, fasting insulin levels were significantly elevated (18.54 ± 4.51 μLU / mL vs. 10.27 ± 2.47 μLU / mL, P = 0.014), and HOMA-IR index and body weight were also significantly increased (P ≤ 0.05). Figure 1 (B, C, D). At 16 weeks, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed. The OGTT results showed no significant difference in fasting blood glucose levels between the two groups. However, the HINS group showed significantly elevated blood glucose levels at 30, 60, 90, 120, 150, and 180 minutes (P≤0.05), and the rate of blood glucose reduction to normal levels was slower. Furthermore, the area under the curve (AUC) was significantly increased in the HINS group (P≤0.05). Figure 1 (E); ITT test results showed that there was no significant difference in blood glucose levels between the two groups of rats at 0 min and 15 min. At 30 min, 45 min, 60 min, 90 min, 120 min, and 150 min, the fasting blood glucose levels in the HINS group were significantly higher than those in the NC group (P≤0.05), and the area under the curve was significantly increased in the HINS group (E). Figure 1 (P < 0.0001). These results indicate that the rat model of hyperinsulinemia was successfully established.

[0060] 1.2.2 HE staining of cardiac tissue

[0061] Studies have shown that hyperinsulinemia can impair cardiac function in mice and rats, causing certain pathological changes. We used male rats fed a high-fat diet for 12 weeks as our research subjects and employed HE staining to detect pathological changes in cardiac tissue between rats fed a high-fat diet for 12 weeks and a control group fed a normal diet. Figure 1 As shown, H&E staining was used to observe the structure of cardiomyocytes in the two groups. In the NC group, cardiomyocytes were spindle-shaped, regularly arranged, and uniformly stained, with no breakage or interstitial cell congestion and proliferation observed. In the HINS group, cardiomyocytes showed a wavy arrangement, with cellular and interstitial edema and vacuolation. Figure 1 (G). This indicates that rats with hyperinsulinemia fed a high-fat diet for 12 weeks exhibited certain pathological changes and can serve as an animal model of myocardial injury in hyperinsulinemia.

[0062] 1.2.3 Immunohistochemical and Western Blotting Results of Cardiac Tissue

[0063] Western and RT-PCR results showed that the expression of AQP1 protein and mRNA in the heart tissue of the HINS group was significantly higher than that of the NC group (p < 0.05). Immunohistochemical results showed that the area of ​​AQP1-positive brown regions in the heart of the HINS group was significantly larger than that in the NC group. Figure 1 (G, H).

[0064] Example 2: In vitro experimental verification of high-concentration insulin-induced expression of AQP1 protein in cardiac cells

[0065] 2.1 Experimental Procedure

[0066] 2.1.1 Cell Culture

[0067] (1) Cell culture conditions are shown in Table 1.

[0068] Table 1: Cell Culture Conditions

[0069]

[0070] Cell growth requires a sterile environment.

[0071] (2) Cell resuscitation

[0072] Prepare the complete culture medium needed for cell culture 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 place them in water at approximately 37°C to thaw (rapid cell freezing solution can damage cells at room temperature). After thawing, on a sterile operating table, transfer the cells to a 15 mL centrifuge tube containing complete culture medium, centrifuge at 1000 rpm / min, balance, and centrifuge for 5 minutes. Discard the supernatant, retain the cell pellet, add 4 mL of complete culture medium, and repeatedly pipette the cells to resuspend them evenly. Then, transfer the suspension to a culture flask, observe under a microscope, and return it to the incubator.

[0073] (3) Change the cell culture medium

[0074] Disinfect hands with alcohol, remove cells from the incubator, and observe growth under a microscope. When changing the culture medium, use a pipette to remove the old medium, add 2 mL of sterile PBS buffer or trypsin to rinse the culture flask to remove dead cells, discard the washing solution, add 4 mL of complete culture medium to each culture flask, and return it to the incubator. Perform aseptic procedures throughout the process.

[0075] (4) Cell passage

[0076] When cells reach a suitable density (70%-80%), passage is necessary to ensure a suitable growth rate. First, disinfect hands, observe cell density, and remove old culture medium with a pipette. Add 2 mL of trypsin to each culture flask to digest the cells, and observe the cell state dynamically under a microscope. When cells lose their normal morphology and detach from the flask wall in a round shape, add 2 mL of complete culture medium (avoid prolonged digestion to prevent cell damage). Then, use a pipette to gently agitate the flask walls to transfer the adherent cells into the complete culture medium and trypsin mixture. Transfer the mixture to centrifuge tubes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and retain the cell pellet. Add an appropriate amount of complete culture medium, gently agitate to distribute the cells evenly in the suspension, and add the pellet to the culture flasks according to the passage plan (maintaining aseptic technique throughout).

[0077] (5) Cell cryopreservation

[0078] First, disinfect your hands and prepare the cell gradient cryopreservation solution on a clean bench. Prepare a solution of 90% fetal bovine serum and 10% cell-specific DMSO at a ratio of 9:1. After removing the cells, centrifuge them according to the passage procedure. After centrifugation, discard the supernatant, add an appropriate amount of cell cryopreservation solution, and then aliquot into cell-specific cryovials for gradient cooling. The next day, store them in liquid nitrogen.

[0079] (6) Cell plating and insulin intervention

[0080] Cell digestion, centrifugation, and passage are performed as described above. Cells are seeded at an appropriate density into six-well plates. When cells reach 70-80% confluence, the old culture medium is discarded, and DMEM high-glucose medium is added to starve the cells for 48 hours. Subsequently, the cells are treated with different concentrations of insulin for 24 hours.

[0081] (7) Cell viability detection

[0082] 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 thoroughly mix with a pipette. 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 further culture; 3) On the second day, after the cells have adhered, discard the old culture medium, add 100 μL of serum-free culture medium to each well to starve the cells for 48 hours, add the intervention solution, and add only PBS to the blank control wells; 4) After the intervention time is over, add 10 μL of CCK8 solution to each well and then return it to the incubator for further incubation; 5) Set the OD value of the microplate reader to 570 mm and detect the readings of the 96-well plate at 30 min, 1 h, 2 h, and 2.5 h.

[0083] 2.1.2 Cell protein extraction

[0084] 1) Prepare 1×PBS 2L; 2) Prepare cell lysis buffer according to the ratio of RIPA:PMSF:phosphatase inhibitor 100μL:1μL:1μL, mix well and set aside; 3) Label EP tubes; 4) After removing cells from the cell culture incubator, discard the old culture medium, wash the cells three times with PBS solution, aspirate the PBS, add 400μL of lysis buffer to each well of a six-well plate, grind thoroughly with a 1mL pipette tip and aspirate the liquid into the EP tube; 5) Centrifuge at 4℃ 3000rpm / min for 5min, transfer the obtained supernatant to the labeled EP tube, record the volume of the supernatant, and store at -80℃.

[0085] 2.1.3 Cellular Immunofluorescence

[0086] 1) First, spread the cells onto a slide; 2) Discard the culture medium, rinse three times with PBS, aspirate the remaining liquid, and fix the cells with cell-specific fixative for 5-10 min; 3) Discard the fixative, rinse three times with PBS for 5 min each time, and aspirate the PBS; 4) Add an appropriate amount of 1% Triton to perforate the cells for 15 min, then rinse the cells with PBS for 3 min each time for 5 min; 5) Block the cells with 1% BSA 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 overnight at 4°C; 7) Equilibrate at 37°C for 30 min the next day, recover the primary antibody (which can be reused), and rinse the cells three times with PBS for 5 min each time; 8) Add an appropriate amount of fluorescent secondary antibody, incubate at 37°C for 30 min, then rinse three times with PBS for 5 min each time; 9) Mount the slide with DAPI containing an anti-quenching agent, stain the cell nuclei, and stain in the dark for 10 min; 10) Take pictures under a microscope and save the images.

[0087] 2.2 Experimental Results

[0088] 2.2.1 CCK8 assay to detect cell viability after intervention with different concentrations of insulin

[0089] Cells were treated with insulin at concentrations of 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 for 24 hours. The results are shown in the figure. Insulin concentrations from 0 ng / mL to 500 ng / mL had virtually no effect on cell viability. However, cell viability decreased to approximately 80% at 1000 ng / mL and only about 70% at 2500 ng / mL. Therefore, subsequent experiments were conducted using an insulin concentration gradient from 0 ng / mL to 500 ng / mL. Figure 2 (C)

[0090] 2.2.2 Effects of different concentrations of insulin on key proteins regulating water, sodium, and calcium ions in H9c2 cells

[0091] H9c2 cardiomyocytes were treated with insulin at concentrations of 0 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 250 ng / mL, and 500 ng / mL. The results are shown in the figure. P-AKT, AQP1, and NHE1 expression were highest at an insulin concentration of 500 ng / mL, and the protein expression levels of P-AKT, AQP1, and NHE1 gradually increased with increasing insulin concentration from 0 ng / mL to 500 mg / mL. Therefore, 500 ng / mL was chosen as the subsequent cell intervention concentration. Figure 2 (D, E).

[0092] 2.2.3 High-concentration insulin intervention increased AQP1 expression in H9c2 cardiomyocytes.

[0093] Western blot analysis showed that, compared with the control group, the expression of AQP1 in cardiomyocytes was significantly increased in the insμLin intervention group. Figure 2 (A, B)

[0094] Example 3: Effects of AQP1 on cardiomyocytes under high insulin concentrations

[0095] 3.1 Experimental Procedure

[0096] 3.1.1 Confocal microscopy stacking scan to detect cell edema

[0097] 1) Turn on the instrument power and light source: First, turn on the microscope and laser, then turn on 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: Locate the cells to be detected under the fluorescence microscope by adjusting the objective lens magnification. 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) Turn off the instrument: After the instrument has finished detecting the sample, first turn off the laser, while the computer can continue image and data processing; 5) Use ZEN software for image processing and analysis.

[0098] 3.1.2 Detection of sodium ion concentration by cellular atomic absorption spectrometry

[0099] 1) Cell suspension collection: Remove cells from the incubator, discard the old culture medium, and wash 2-3 times with sterile PBS. Add 2 mL of EDTA to each well of a 6-well plate, incubate at 37°C for 3 min, then remove and add 2 mL of complete culture medium to stop digestion. Collect the EDTA-culture medium mixture into a 4 mL EP tube, label each well as its group, and then centrifuge at 1000 rpm for 5 min to collect the cell pellet. Add approximately 200 μL of sterile PBS to each cell pellet (the appropriate amount of PBS depends on the number of cells), and sonicate the cells three times for 2 min each time (sonicator settings: 20 Hz). After centrifugation and disruption of the cell slurry, transfer the supernatant to a new 2mL ep tube; 2) Preparation of standard solutions: Dilute the standard solutions to five concentrations of 8, 4, 2, 1, and 0.5mM respectively, and set aside; 3) Sample loading: Add 100μL of the standard solutions and the prepared samples to the 96-well plate respectively, plot the standard curve based on the absorbance of different wells, and calculate the final concentration of each sample.

[0100] 3.1.3 Fluo-4AM Calcium Ion Fluorescent Probe Detection

[0101] 1) Take an appropriate amount of Fluo-4AM stock solution and dilute it with PBS to a working solution of 2 WM. Note: The working solution must be prepared immediately before use and should not be repeatedly frozen and thawed; 2) After removing the culture medium from the adherent cells, wash them three times with PBS. Note: Because the serum in the culture medium contains esterase, which will cause Fluo-4AM to decompose into Fluo-4, and phenol red will cause enhanced fluorescence background; 3) Add Fluo-4AM working solution, the volume of which should be sufficient to fully cover the cells. For a six-well plate, 1 mL is generally sufficient; 4) Incubate at 37℃ for 30 min to load the fluorescent probe. Note: If this is the 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. If the fluorescence is too weak, extend the incubation time. 5) Wash with PBS three times. After washing, you can consider incubating for another 20-30 minutes to ensure that Fluo-4AM is completely converted into 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 the changes in intracellular calcium ion concentration.

[0102] 3.1.4 Detection of RHOD-2AM Mitochondrial Fluorescent Probes

[0103] 1) Take an appropriate amount of Rhod-2AM stock solution and dilute it with PBS to 1 μM 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 adherent cells and wash them three times with PBS; 3) Add Rhod-2AM working solution, the volume of which should be sufficient to fully cover the cells. For a six-well plate, 1 mL is generally sufficient; 4) Incubate at 37℃ for 30 min to load the fluorescent probe; 5) Then wash three times with PBS; 6) Use a laser confocal microscope to detect the red specific fluorescence intensity of Rhod-2AM in the cells to determine the calcium ion concentration in the mitochondria.

[0104] 3.1.5 Detection of mitochondrial membrane potential in JC-1 cells

[0105] 1) First, prepare the positive control working solution by diluting it at a ratio of 1:1000 and adding it to the cells. Incubate in an incubator for about 30 minutes. 2) Then, prepare the JC-1 staining working solution by adding 50 μL of JC-1 staining working solution (200X) to 8 mL of ddH2O, mixing well, and then adding 2 mL of JC-1 staining buffer (5X) and mixing thoroughly. 3) After the positive control working solution has incubated in the cell culture incubator, wash the cells three times with PBS for 5 minutes each time. 4) Add 500 μL of complete cell culture medium to each well. 5) Then, add 500 μL of JC-1 staining working solution to each well and incubate in a 37°C incubator for 30 minutes. 6) The JC-1 staining buffer working solution is prepared at a ratio of 1 mL staining buffer (5×): 4 mL... Prepare the ddH2O solution according to the specified ratio (it must be freshly prepared and used immediately); 7) After incubation at 37°C, discard the supernatant of the JC-1 staining working solution and wash twice with the prepared JC-1 staining buffer; 8) Add 2 mL of cell culture medium to each well; 9) Take pictures using a fluorescence microscope and save them.

[0106] 3.2 Experimental Results

[0107] 3.2.1 High-concentration insulin intervention resulted in cell edema in H9c2 cardiomyocytes.

[0108] CalceinAM (calcein acetoxymethyl ester) is a green fluorescent probe with strong hydrophobicity. It penetrates the cell membrane and enters the cell, emitting strong green fluorescence for specific fluorescent labeling. Confocal microscopy stacked scanning results showed that the high-concentration insulin intervention group had an increased number of cell stacked layers, and the cross-sectional area of ​​the brightest cell layer also increased, suggesting an increase in cell volume. Figure 3 (A)

[0109] 3.2.2 Abnormal expression of sodium and calcium regulatory proteins after high-concentration insulin intervention in H9c2 cardiomyocytes

[0110] Western blot results showed that, compared with the control group, the high-concentration insulin intervention group had significantly increased levels of NHE1 and SGLT1 proteins, while the levels of NKA, NCX, and SERCA proteins decreased. Figure 3 (B)

[0111] 3.2.3 High-concentration insulin intervention induces sodium-calcium overload in H9c2 cardiomyocytes

[0112] Fluo-4 AM (Fluo-4 Calcium) is a specific fluorescent probe for calcium ions. Fluo-4 AM itself is almost non-fluorescent, but once it enters the cell, it is hydrolyzed by intracellular esterases, producing Fluo-4, which binds to calcium ions and emits green fluorescence. Confocal microscopy results showed that, compared with the control group, the Fluo-4 AM signal was increased in the insulin intervention group. Atomic absorption spectrometry results showed that, compared with the control group, the absorbance of sodium ions was increased in the insulin intervention group. Figure 3 C, Figure 4 (A)

[0113] 3.2.4 Insufficient mitochondrial calcium content after high-concentration insulin intervention in H9c2 cardiomyocytes

[0114] Rhod-2AM is a specific mitochondrial fluorescent probe that effectively increases mitochondrial calcium fluorescence signal by 80-100 times, exhibiting the strongest signal intensity among all mitochondrial calcium ion probes. Therefore, Rhod-2AM is commonly used as a specific probe to detect intramitochondrial calcium ion levels. Confocal microscopy results showed that, compared to the control group, the high-concentration insulin intervention group exhibited weakened Rhod-2AM signal in mitochondria, while ROS expression in the cells increased. Figure 4 (B)

[0115] 3.2.5 High concentrations of insulin induce mitochondrial dysfunction in H9c2 cardiomyocytes

[0116] Western blot results showed that, compared with the control group, the expression of PGC1α and OPA1 proteins was significantly reduced in the insulin intervention group, while the p-DRP1 / DRP1 protein level was significantly increased.

[0117] 3.2.6 High concentrations of insulin induced damage to mitochondrial membrane potential in H9c2 cardiomyocytes

[0118] 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 a polymer and producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix, and remains in monomeric form, producing green fluorescence. The transition from red to green fluorescence of JC-1 is usually used as an indicator of a decrease in cell membrane potential. Compared to the control group, the high-concentration insulin intervention group showed enhanced green fluorescence, decreased red fluorescence, and a lower mitochondrial membrane potential. Figure 4 (C)

[0119] Example 4: siRNA-AQP1 improves high-insulin-induced cardiomyocyte injury

[0120] 4.1 Experimental Procedure

[0121] 4.1.1 Intracellular transfection of H9c2 cells with si-AQP1

[0122] 1) Seed H9c2 cells into 6-well plates at a density of 5 × 10⁸ cells per well, and transfect the cells the next day; 2) Prepare serum-containing medium without antibiotics; 3) Remove the old medium from the 6-well plates and wash the cells twice with PBS; 4) Prepare a mixture by adding 245 μL of Opti-Free and 5 μL of Lipofectamine 3000 to each well of the 6-well plate, mix well, and incubate at room temperature for 5 min; 5) Dilute the AQP1 inhibitor negative control and AQP1 inhibitor to working concentrations with 250 μL of DEPC water according to the manufacturer's instructions. Similarly, prepare the mixture by adding 245 μL of OPTI and 5 μL of AQP1 inhibitor to each well of a 6-well plate, mix well by pipetting, and incubate at room temperature for 5 min. 6) Mix the diluted Lipo 3000-OPTI mixture and the diluted inhibitor-OPTI mixture, and incubate at room temperature for 20 min to prepare the AQP1 inhibitor-lipo3000 complex. 7) Discard the old culture medium in the 6-well plate, wash the cells twice with PBS, add 1.5 mL of OPTI solution to each well, then add 500 μL of the AQP1 inhibitor-lipo3000 complex, adding it dropwise around the edge. Add 2 mL of OPTI solution to the blank control well. 8) Place the 6-well plate back into the cell culture incubator, dynamically observe the cell status, and after incubation for 4-6 h, aspirate the old culture medium and replace it with 2 mL of medium without antibiotics, and culture for 48 h. 9) After the intervention period, extract the cell proteins from each well.

[0123] 4.2 Experimental Results

[0124] 4.2.1 Validation of the si-AQP1 knockdown sequence

[0125] Western blot results showed that 48 h after si-AQP1 transfection, the expression of AQP1 protein in the AQP1-siRNA-2 sequence decreased most significantly, by approximately 73% compared to the ctlsiRNA group, a statistically significant difference (P < 0.05). Cy5 is a red fluorescent probe capable of specifically labeling knockdown sequences. After staining cells with the Cy5 fluorescent probe, confocal microscopy revealed that the specific red fluorescence of Cy5 successfully fused with the green fluorescence used to stain the cytoplasm. These results indicate that the intracellular si-AQP1 knockdown sequence was successfully constructed. Figure 5 (A, B)

[0126] The si-AQP1 target sequence is:

[0127] GCAGCAGAGTAAAGGTCAT.

[0128] 4.2.2 Insulin intervention reduces cellular edema with si-AQP1

[0129] Next, the cell edema after si-AQP1 intervention with ins was examined. Confocal microscopy results showed that, compared to the ins intervention group, the number of cell layers scanned in the ins+si-AQP1 group decreased from 19 to 13, and the cross-sectional area of ​​the brightest cell layer was also significantly reduced. Figure 5 (C)

[0130] 4.2.3 Under insulin intervention, si-AQP1 can alleviate cellular water, sodium, and calcium ion imbalance.

[0131] Next, Western blot analysis was used to detect key regulatory proteins of water and sodium-calcium balance. The results showed that compared with the ins+si-AQP1 group, the expression levels of NHE1 and SGLT1 proteins, which transport sodium ions into cells, were significantly decreased in the ins+si-AQP1 group (P < 0.05), while the expression levels of NKA, SERCA, and NCX proteins, which expel sodium and calcium ions from the cell, were increased (P < 0.05). The expression levels of key mitochondrial biosynthesis proteins PGC1α, mitochondrial fusion proteins MFN1 and OPA1 were significantly increased, while the expression of mitochondrial splitting protein DRP1 was decreased. Figure 6 (A)

[0132] 4.2.4 Insulin intervention reduces cellular sodium and calcium overload and mitochondrial calcium deficiency.

[0133] Further observation of sodium and calcium ion concentrations in each group of cells was conducted. Confocal microscopy results showed that, compared with the insulin intervention group, the expression of the green fluorescent signal FLUO-4AM of calcium ions in the cytoplasm of cardiomyocytes decreased in the ins+si-AQP1 group, and the sodium ion concentration in cells was decreased as detected by atomic absorption spectrometry (P < 0.05); the expression of the red fluorescent signal RHOD-2AM of calcium ions in mitochondria increased (P < 0.05). Figure 6 (B)

[0134] 4.2.5 Insulin intervention reduces the decrease in si-AQP1 mitochondrial membrane potential in cardiomyocytes

[0135] JC-1 mitochondrial membrane potential staining results showed that, compared with the ins intervention group, the ins+si-AQP1 group showed enhanced red fluorescence and decreased green fluorescence, indicating an increased mitochondrial membrane potential. Figure 7 ).

[0136] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. The application of siRNA in the preparation of drugs for treating myocardial injury in patients with hyperinsulinemia, characterized in that, The siRNA inhibits the expression of AQP1; The target sequence of the siRNA is: GCAGCAGAGTAAAGGTCAT.

2. The application according to claim 1, characterized in that, The hyperinsulinemia-related myocardial injury includes any one of cardiomyocyte edema, sodium and calcium ion overload, or mitochondrial dysfunction.

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