Application of lysophosphatidyl ethanolamine in preparation of medicine for treating hepatocyte and / or myocardial cell injury caused by hyperuricemia

Abstract

The invention discloses application of lysophosphatidyl ethanolamine in preparation of a medicine for treating hepatocyte and / or myocardial cell injury caused by hyperuricemia, and belongs to the field of medicine preparation. According to the application disclosed by the invention, whether hypeluricemia induces hepatocyte ferroptosis or not and whether lysophosphatidyl ethanolamine has an improvement effect on hepatocyte and myocardial cell injury induced by hypeluricemia or not are researched, and results show that hypeluricemia can induce hepatocyte ferroptosis; the lysophosphatidyl ethanolamine can be used for treating hepatocyte injury by improving hepatocyte mitochondrial injury, improving hepatocyte lipid peroxidation and improving hepatocyte iron overload and oxidative stress conditions caused by hyperuricemia. The lysophosphatidyl ethanolamine is used for treating myocardial cell injury by reducing myocardial cell apoptosis and improving myocardial cell mitochondrial injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation, specifically the application of lysophosphatidylethanolamine (LPE) in the preparation of drugs for treating hepatocyte and / or myocardial cell damage caused by hyperuricemia. Background Technology

[0002] Hyperuricemia has become the second most common metabolic disease after diabetes, but its clinical management strategies remain highly controversial globally. On the one hand, the "Chinese Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout (2019)" recommends that for asymptomatic patients, the threshold for uric acid-lowering drug therapy is a serum uric acid level > 540 μmol / L, far exceeding the definition value of hyperuricemia (420 μmol / L). On the other hand, European and American guidelines do not recommend drug intervention in the absence of complications. This discrepancy highlights the current lack of understanding of the pathogenic role of hyperuricemia. Meanwhile, how uric acid (UA), as an antioxidant, induces multi-system diseases is also a key scientific question that urgently needs to be elucidated. Therefore, a deeper understanding of the pathogenic mechanism of hyperuricemia is of urgent practical significance for unifying clinical practice and developing precise prevention and treatment strategies.

[0003] Hyperuricemia is not only a major cause of gout, but also an independent risk factor for metabolic syndrome, type 2 diabetes, cardiovascular and cerebrovascular diseases, hypertension, and chronic kidney disease. In the liver, xanthine is converted into uric acid under the catalysis of xanthine oxidase. Given that the liver is the organ with the highest expression of this enzyme, hepatocytes are continuously exposed to a high concentration of uric acid, making it a potential target for hyperuricemia-induced damage. Existing studies have confirmed that hyperuricemia participates in the pathological processes of non-alcoholic fatty liver disease, alcoholic fatty liver disease, and liver fibrosis by inducing oxidative stress, endoplasmic reticulum stress, inflammatory responses, and apoptosis. However, the specific molecular mechanisms by which it causes liver damage are not yet fully elucidated. Notably, the association between hyperuricemia and ferroptosis has begun to attract attention in recent years. Studies have found that hyperuricemic mice exhibit kidney damage manifestations such as elevated serum creatinine and blood urea nitrogen, accompanied by downregulation of GPX4 and xCT expression. The ferroptosis inhibitor Ferrostatin-1 (Fer-1) can improve kidney damage, inflammatory cell infiltration, and fibrosis in mice. In addition, high uric acid can promote cardiomyocyte ferroptosis by increasing oxidative stress and downregulating GPX4. However, whether high uric acid can induce hepatocyte ferroptosis has not been reported, and the mechanism is unknown.

[0004] Iron metabolism imbalance is a key factor in ferroptosis. After dietary iron is absorbed in the duodenum, it carries Fe... 3+ After transferrin (TF) binds to the transferrin receptor (TFR1, also known as CD71 or TFRC) on the plasma membrane, the plasma membrane forms endosomes that carry Fe. 3+TF is taken into cells, and Fe in the endosomes 3+ Reduced to ferrous ions (Fe) by the metalloreductase STEAP3 2 + Subsequently, Fe is released into the cytoplasm via DMT1 (divalent metal ion transporter 1). 2+ It is used or stored in the unstable iron pool (LIP) and ferritin. Excessive free iron enters the mitochondria, affecting the synthesis of Fe-S (iron-sulfur clusters). The iron metabolism, mitochondrial respiration, and redox balance involved in Fe-S are impaired, leading to mitochondrial dysfunction, which is morphologically manifested as smaller mitochondrial volume, increased membrane density, and a decrease or disappearance of cristae.

[0005] Mitochondrial functional integrity is the cornerstone of cell survival. Phosphatidylethanolamine (PE), as the main phospholipid component of the mitochondrial membrane (accounting for approximately 30%), is crucial for maintaining mitochondrial structure and function, and is deeply involved in regulating key life activities such as apoptosis, energy metabolism, and autophagy. In mammalian cells, PE synthesis is mainly accomplished through four pathways, and the PE produced by each pathway has independent physiological functions. Among them, the phosphatidylserine decarboxylase (PISD) pathway is the most important source of endogenous mitochondrial PE synthesis. Studies have shown that PISD is essential for mouse embryonic development; knocking out PISD in mice reduces the PE content in mitochondria, leading to mitochondrial morphological changes and embryonic death. Therefore, mitochondrial PISD plays a very important role in hepatocyte injury. However, in the specific pathological context of hyperuricemia, whether and how lysophosphatidylethanolamine (LPE) participates in regulating mitochondrial function in cardiomyocytes remains unexplored to date.

[0006] In summary, there are two key scientific questions that urgently need to be addressed in the current research field: First, does hyperuricemia induce hepatocyte ferroptosis? Second, does LPE have a mitigating effect on hyperuricemia-induced hepatocyte and cardiomyocyte damage? To address these gaps, this invention aims to systematically study the mitigating effects of exogenous LPE on these two issues, thereby providing a novel theoretical basis and potential intervention strategies for the prevention and treatment of hyperuricemia-related liver injury and cardiac dysfunction. Summary of the Invention

[0007] This invention provides the use of lysophosphatidylethanolamine in the preparation of drugs for treating hepatocellular and / or myocardial cell damage caused by hyperuricemia.

[0008] Firstly, this invention demonstrates through specific embodiments that lysophosphatidylethanolamine has a therapeutic effect on hepatocellular damage caused by hyperuricemia.

[0009] (1) The lysophosphatidylethanolamine described herein treats hepatocellular damage by improving mitochondrial damage and lipid peroxidation in hepatocellular cells. Specifically:

[0010] The lysophosphatidylethanolamine increases mitochondrial membrane potential, improves hepatocyte mitochondrial damage, and thus treats hepatocyte damage.

[0011] The lysophosphatidylethanolamine treats hepatocellular damage by reducing malondialdehyde (MDA) and lipid peroxide (LPO) levels, thereby improving hepatocellular lipid peroxidation.

[0012] (2) The lysophosphatidylethanolamine treats hepatocellular damage by improving hepatocellular iron overload and oxidative stress caused by hyperuricemia. Specifically:

[0013] The lysophosphatidylethanolamine works by reducing Fe in hepatocytes. 2+ The level improves hepatocyte iron overload caused by high uric acid, thereby treating hepatocyte damage;

[0014] The lysophosphatidylethanolamine treats hepatocellular damage by reducing the accumulation of reactive oxygen species in hepatocytes and improving oxidative stress caused by hyperuricemia.

[0015] The lysophosphatidylethanolamine improves oxidative stress associated with mitochondrial dysfunction caused by hyperuricemia by reducing the expression levels of NOX4 and HIF2α proteins, thereby treating hepatocellular damage.

[0016] Secondly, through specific embodiments, this invention demonstrates that lysophosphatidylethanolamine has the effect of treating myocardial cell damage caused by hyperuricemia.

[0017] The lysophosphatidylethanolamine treats cardiomyocyte damage by reducing cardiomyocyte apoptosis and improving mitochondrial damage in cardiomyocytes. Specifically:

[0018] The lysophosphatidylethanolamine reduces cardiomyocyte apoptosis by increasing Bcl-2 protein levels and decreasing BAX and cleaved Caspase-3 protein levels.

[0019] The lysophosphatidylethanolamine treats myocardial cell damage by reducing the accumulation of reactive oxygen species in myocardial hepatocytes and improving oxidative stress caused by hyperuricemia.

[0020] The lysophosphatidylethanolamine improves mitochondrial damage in cardiomyocytes by reducing mitochondrial membrane potential, enhancing mitochondrial respiratory function, and reducing mitochondrial structural damage, thereby treating cardiomyocyte damage.

[0021] Beneficial effects: This invention demonstrates through specific embodiments that lysophosphatidylethanolamine has the effect of treating hepatocyte and / or myocardial cell damage caused by hyperuricemia. Attached Figure Description

[0022] Figure 1 shows the results of detecting iron overload, oxidative stress, and lipid peroxidation in hepatocytes induced by hyperuricemia; where A is the proliferation curve of L02 cells; B is the proliferation curve of THLE-2 cells; C is the effect of hyperuricemia on ROS content in THLE-2 hepatocytes detected by the DCFH-DA probe (n=3); D is the effect of hyperuricemia on ROS content in THLE-2 hepatocytes detected by flow cytometry (n=3); (E) is the Western spectral analysis. Blot analysis of NOX4 protein levels in THLE-2 hepatocytes (n=3); (F) Effect of the kit on the Fe²⁺ content in THLE-2 hepatocytes (n=3); G) LPO level in THLE-2 hepatocytes (n=3); H) 4-HNE level in THLE-2 hepatocytes (n=3); I) MDA level in THLE-2 hepatocytes (n=3); (J) Effect of the JC-1 probe on the mitochondrial membrane potential of THLE-2 hepatocytes (n=3); *P<0.05, **P<0.01.

[0023] Figure 2 The figure shows the detection results of LPE on ferroptosis induced by high uric acid in hepatocytes: where A represents the Fe content of THLE-2 hepatocytes after LPE pretreatment as detected by the kit. 2+ Levels (n=3); B: Reactive oxygen species (ROS) level in LPE-pretreated hepatocytes L02 detected by DCFH-DA probe (n=3); C: Effect of high uric acid on ROS content in L02 hepatocytes detected by flow cytometry (n=3); D: Protein levels of NOX4 and HIF-2α in LPE-pretreated THLE-2 hepatocytes under HUA detected by Western blotting (n=3); E: Mitochondrial membrane potential level in SKQ1-pretreated THLE-2 hepatocytes detected by JC-1 probe (n=3); F: LPO level in SKQ1-pretreated THLE-2 hepatocytes (n=3); G: MDA level in SKQ1-pretreated THLE-2 hepatocytes (n=3); *P<0.05, **P<0.01, relative to the control group.

[0024] Figure 3 shows the results of high uric acid-induced mitochondrial dysfunction, enhanced oxidative stress, and apoptosis in AC16 cardiomyocytes. A represents the expression levels of Bax, cleaved Caspase-3, and Bcl-2 proteins in AC16 cardiomyocytes after HUA treatment, detected by Western blotting (n=3); B represents the apoptosis of AC16 cardiomyocytes in the control group and the HUA model group, detected by flow cytometry; C represents the intracellular reactive oxygen species (ROS) levels in cardiomyocytes of each group, assessed using the DCFH-DA probe (n=3); D represents the effect of HUA on the real-time mitochondrial oxygen consumption rate (OCR) of AC16 cells (n=3); E represents the mitochondrial membrane potential of each group, detected using the JC-1 probe (n=3). *p < 0.05, **p < 0.01.

[0025] Figure 4 shows the results of LPE reversing hyperuricemia (HUA)-induced mitochondrial dysfunction, oxidative stress, and apoptosis in AC16 cardiomyocytes. A shows the expression levels of Bax, cleaved Caspase-3, and Bcl-2 in AC16 cardiomyocytes after LPE treatment under HUA (n=3); B shows the reactive oxygen species (ROS) levels in AC16 cardiomyocytes of each group detected by the DCFH-DA probe method (n=3); C shows the mitochondrial membrane potential levels in AC16 cardiomyocytes of each group detected by the JC-1 probe method (n=3); D shows the real-time detection of mitochondrial oxygen consumption rate in AC16 cardiomyocytes of each group by the Seahorse mitochondrial stress test (n=3); E shows the ultrastructural changes of mitochondria in AC16 cardiomyocytes observed by transmission electron microscopy; *p<0.05, **p<0.01. Detailed Implementation

[0026] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0027] Example 1: The effect of exogenous lysophosphatidylethanolamine on improving ferroptosis in hepatocytes induced by hyperuricemia.

[0028] 1. Main reagents, consumables, and main instruments used in the experiment:

[0029] Table 1. Main reagents and consumables used in the experiment

[0030]

[0031] Table 2. Main Experimental Instruments

[0032]

[0033] 2. Cells, cell culture, and cell models:

[0034] (1) Immortalized hepatocytes THLE-2 (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin / 100 μg / mL streptomycin; hepatocytes L02 (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin / 100 μg / mL streptomycin. All cells were cultured in an incubator at 37°C with 5% CO2.

[0035] (2) To establish an in vitro model of hyperuricemia (HUA), the uric acid solution was prepared as follows: 0.0252 g of uric acid was added to 3 mL of 1 M NaOH solution, vortexed to dissolve it completely, and then concentrated hydrochloric acid was added to adjust the pH to 7.2-7.4 to prepare a 50 mM uric acid stock solution. The uric acid stock solution was filtered through a 0.22 μm filter membrane and diluted with appropriate DMEM culture medium to a concentration of 750 μmol / L uric acid solution.

[0036] After the uric acid solution was prepared, THLE-2 cells and L02 cells were cultured in a medium containing 750 μmol / L UA for 48 h to establish in vitro HUA models of THLE-2 cells and L02 cells, respectively.

[0037] (3) In the mitochondrial antioxidant (SKQ1) pretreatment experiment, 500 nmol / L SKQ1 was added to the cells, and 750 μmol / L UA was added to stimulate for 48h.

[0038] (4) In the lysophosphatidylethanolamine (LPE) pretreatment experiment, 100 μmol / L LPE was added to the cells, and 750 μmol / L UA was added to stimulate for 48 h.

[0039] 3. Detection of reactive oxygen species (ROS) in cells:

[0040] The working solution of DCFH-DA (a fluorescent probe for detecting intracellular reactive oxygen species (ROS)) was diluted to 10 μmol / L according to the ratio of serum-free culture medium to DCFH-DA = 1000:1 (v / v). Cells were seeded in six-well plates. After drug administration and modeling, the cells were washed twice with PBS, and 1 mL of working solution was added. The cells were incubated in an incubator for 30 min. After incubation, the cells were washed three times with 1640 basal medium. Finally, 2 mL of PBS was added for sealing. The ROS level was quantitatively analyzed using a fluorescence microscope.

[0041] 4. Detection of mitochondrial membrane potential:

[0042] Dilute JC-1 to 8 mL of ultrapure water at a ratio of 50 μL JC-1 (200×), vortex to mix, and then add 2 mL of 5× JC-1 staining buffer to prepare the JC-1 staining working solution. Seed cells in six-well plates, and after drug administration and cell modeling, wash twice with PBS. Mix the JC-1 staining working solution with an equal volume of 1640 complete culture medium, add 2 mL to each well of the six-well plate, and incubate for 30 min. Dilute the JC-1 staining buffer to ultrapure water at a ratio of 5× JC-1: 1:5 (v / v) and place on ice. After cell incubation, wash twice with JC-1 buffer, add 2 mL of 1640 culture medium, and observe under a fluorescence microscope.

[0043] 5. Detection of cellular LPO (lipid peroxide) levels:

[0044] (1) Reagent preparation: Before testing, remove the reagents from the kit and allow them to return to room temperature; mix the stock solution and diluent at a ratio of 3:1 (v / v) to prepare the LPO working solution. Dilute the 100 μmol / L standard to different concentrations according to Table 3.

[0045] Table 3. Dilution Table of LPO Standard Concentration

[0046]

[0047] (2) Sample preparation: Seed cells in six-well plates. After drug administration and modeling, wash twice with PBS, add 300 μL of pre-cooled PBS, and place in an ultrasonic disruptor. After cell disruption, centrifuge at 4°C and 12000×g for 10 min, and collect the supernatant.

[0048] (3) Operating steps:

[0049] Take 200 μL of standard or test sample of different concentrations and add it to the corresponding EP tube;

[0050] Add 650 μL of LPO working solution to each of the above tubes, mix thoroughly by blowing, and then add 150 μL of acid solution to each tube.

[0051] The mixture was reacted in a 45°C water bath for 60 min, and then cooled to room temperature by running water. Each tube was centrifuged at 1100×g for 10 min.

[0052] Add 200 μL of the supernatant to a 96-well plate;

[0053] The OD values ​​of each well were measured at 586 nm using an ELISA reader, and the LPO levels in mouse liver were calculated by substituting the values ​​into the standard curve.

[0054] 6. Detection of cellular MDA (malondialdehyde) levels:

[0055] (1) Sample preparation: Cells were seeded in six-well plates. After drug administration and modeling, the cells were washed twice with PBS, and 200 μL of lysis buffer was added. The plates were then placed on ice for 30 min. Cells were scraped off and transferred to EP tubes. The plates were centrifuged at 12000 × g for 10 min at 4 °C, and the supernatant was collected. The total protein content of the cells was determined using a BCA kit, and the MDA content was subsequently determined.

[0056] (2) Preparation of reagents:

[0057] Preparation of TBA stock solution: Add 6.76 mL of the preparation solution to 0.025 g of TBA to prepare a 0.37% TBA stock solution. A small amount of precipitate will form at this point. Heat the mixture to 70ºC and vortex vigorously to promote dissolution. Store the prepared TBA stock solution at room temperature, protected from light.

[0058] Preparation of MDA working solution: Prepare the working solution according to the ratio of diluent: stock solution: antioxidant = 150μL: 50μL: 3μL for each sample. The working solution should be prepared and used immediately. The MDA working solution also needs to be heated to 70ºC and shaken vigorously to promote dissolution.

[0059] Dilution of standard: Dilute 1 mM MDA standard according to Table 4.

[0060] Table 4. MDA Standard Concentration Dilution Table

[0061]

[0062] (3) Sample testing: Set up the testing reaction system according to Table 5;

[0063] Table 5. MDA Detection System

[0064]

[0065] After mixing all components in the detection system, heat in a boiling water bath at 100ºC for 15 min; cool the sample to room temperature in a cold water bath, centrifuge at 1000×g for 10 min, add 200μL of supernatant to a 96-well plate, and measure the absorbance at 532nm; establish a standard curve to calculate the MDA level in the sample.

[0066] 7. Western blot detection of cell-related protein levels:

[0067] (1) Sample extraction: Inoculate cells in small dishes, after drug administration and modeling, rinse with PBS, add 150 μL of lysis buffer, and incubate on ice for 30 min. Collect cells with a cell scraper, transfer to EP tubes, centrifuge at 4℃ and 12000 rpm for 10 min, and the supernatant is the total cell protein.

[0068] (2) Determination of total protein concentration by BCA method

[0069] Protein concentration was determined using the BCA protein concentration assay kit. BCA reagent A (BCA reagent) and reagent B (copper reagent) were mixed at a volume ratio of 50:1 to prepare the BCA working solution, ensuring thorough mixing. Samples were added according to the instructions in Table 6 to determine the protein concentration.

[0070] Table 6. Protein concentration determination by BCA method

[0071]

[0072] (3) Protein denaturation: Mix the sample with the buffer solution according to the ratio of 5× to protein sample = 4:1, and then denature the protein in a metal bath at 100°C for 10 min. Cool to room temperature and then electrophoresis can be performed or stored at -20°C for later use.

[0073] (4) Preparation of SDS-PAGE gel:

[0074] Separating gels of different concentrations were prepared according to Table 7, and 3 mL of isopropanol was added for pressing. After 30 min of treatment, the isopropanol was poured out and the moisture was absorbed with filter paper.

[0075] Table 7. Preparation of separating gel

[0076]

[0077] Prepare the stacking gel according to the reagents in Table 8 below. First, inject the prepared stacking gel liquid into the glass plate with a 1 mL pipette, then slowly insert it into the comb and wait for it to solidify (about 30 min). Then, put the glass plate into the electrophoresis tank, ensuring that the electrophoresis solution is completely filled, and remove the comb for subsequent experiments.

[0078] Table 8. Preparation of stacking gel

[0079]

[0080] (5) Western Blot electrophoresis: Add protein samples and markers to wells (30 μg~50 μg per well), set the voltage to 70 V, and wait until the molecular weight band of the marker is clear (30 min~40 min). Then, turn the voltage to 110 V and continue electrophoresis. Stop electrophoresis immediately when the protein sample is about to reach the bottom of the gel.

[0081] (6) Western Blot Transfer: Cut the PVDF membrane to the required size beforehand and mark it. Then, activate the PVDF membrane in methanol. Next, take out the transfer clamp, sponge pad, and filter paper and place them in the tray in sequence. Pour the transfer buffer into the tray. According to the molecular weight of the target protein, cut the gel and place it on the filter paper. Then, neatly cover the gel with the PVDF membrane, ensuring that no air bubbles are formed during the process of covering the gel with the PVDF membrane. Finally, cover the filter paper and sponge pad in sequence, then clamp the transfer clamp and gently place it into the transfer tank. After completely filling the tank with transfer buffer, place it in an ice bath and maintain a constant current of 200 mA for transfer. Select the appropriate transfer time according to the molecular weight of the protein.

[0082] (7) Sealing: After the transfer is completed, immediately take the PVDF membrane out of the transfer tank and place it in the pre-prepared sealing solution for 1 h to 2 h.

[0083] (8) Primary and secondary antibody incubation: After the blocking is completed, the PVDF membrane is taken out of the blocking solution and washed with TBST 3 times, 5 min each time. After washing, the PVDF membrane surface is dried with TBST using washing paper. Then the PVDF membrane is placed in the corresponding primary antibody and incubated overnight at 4°C. The next day, it is washed with TBST 3 times, 10 min each time. Then the membrane is placed in the secondary antibody box and incubated slowly at room temperature for 1 h. After the incubation is completed, it is washed with TBST 3 times, 10 min each time.

[0084] (9) Development:

[0085] In a light-protected environment, the developing solution was prepared according to a volume ratio of enhanced luminescent liquid to stabilizer of 1:1. After blotting the liquid off the surface of the PVDF film with filter paper, the film was evenly coated with the developing solution on the developing machine, and then exposed in the developing machine to preserve the image. ImageJ software was used for precise quantitative analysis of the grayscale values.

[0086] 8. Experimental Results:

[0087] (1) High uric acid induces iron overload, oxidative stress and lipid peroxidation in hepatocytes.

[0088] To evaluate the effects of high uric acid stimulation on hepatocytes, this study used human normal hepatocytes L02 and immortalized hepatocytes THLE-2 as a parallel two-cell model. The CCK-8 assay was used to continuously monitor cell proliferation dynamics from 0 to 72 h after treatment with 750 μmol / L UA. The results showed that the proliferation activity of both cell lines decreased progressively and continuously with prolonged exposure time, and the inhibitory effect reached statistical significance at 48 h, further intensifying at 72 h, demonstrating a clear time-dependent characteristic. Figure 1(As shown in Figures A and B). The above results established the in vitro modeling conditions for hepatocytes treated with 750 μmol / L UA for 48 h.

[0089] The experiment used the DCFH-DA fluorescent probe combined with flow cytometry to quantitatively analyze intracellular ROS levels. Microscopic imaging showed that the green fluorescence signal in hepatocytes of the HUA-treated group was significantly enhanced compared with that of the control group (hepatocytes of the untreated group), indicating a significant increase in ROS levels. Figure 1 (C); Flow cytometry further confirmed the significant difference in fluorescence intensity between the two groups ( Figure 1 The results showed that HUA can induce oxidative stress in hepatocytes. Western blotting analysis revealed that the expression of NOX4, a core protein of oxidative stress, was significantly upregulated after HUA stimulation. Figure 1 The changes in HUA (e.g., E) are consistent with those in ROS levels, indicating that HUA can activate the NADPH oxidase system and amplify cellular oxidative damage. (The text then abruptly shifts to a seemingly unrelated topic about Fe...) 2+ The test kit results showed that the Fe²⁺ content in the HUA-treated group was significantly higher than that in the control group. Figure 1 (F), confirming that HUA can cause a significant expansion of the intracellular unstable iron pool.

[0090] Three lipid peroxidation markers were detected using a assay kit: total lipid peroxides (LPO), the electrophilic aldehyde product 4-hydroxynonenal (4-HNE), and the lipid terminal degradation product malondialdehyde (MDA). The results showed that, compared with the control group, the levels of LPO, 4-HNE, and MDA in hepatocytes of the HUA-treated group were significantly increased. Figure 1 The study confirmed extensive oxidative damage to cell membrane phospholipids using the JC-1 fluorescent probe. Mitochondrial membrane potential was assessed using the JC-1 fluorescent probe, and the results showed a significant decrease in mitochondrial membrane potential in the HUA-treated group compared to the control group. Figure 1 (J), indicating that mitochondria are depolarizing and their function is impaired.

[0091] (2) Exogenous lysophosphatidylethanolamine can improve the iron overload and oxidative stress of hepatocytes caused by hyperuricemia.

[0092] To verify that mitochondrial dysfunction in hepatocytes caused by hyperuricemia (HUA) is indeed related to a decrease in mitochondrial phospholipids (mtPE), this study treated cells with 100 μM LPE, primarily to promote mtPE synthesis. After 48 hours of HUA treatment, cells supplemented with LPE showed an increase in mtPE levels.

[0093] Using a kit for detection, the results showed that compared with the control group (hepatocytes not treated with HUA), hepatocytes stimulated by high uric acid had higher Fe content. 2+The levels increased. However, after LPE pretreatment, Fe in hepatocytes... 2+ The level decreased significantly ( Figure 2 (A). The results of reactive oxygen species (ROS) detection showed that, compared with the control group, high-concentration uric acid stimulation led to increased accumulation of ROS in hepatocytes, while LPE pretreatment effectively reduced this ROS accumulation induced by high uric acid. Figure 2 (BC). Furthermore, the expression of NOX4 and HIF2α in the HUA group was significantly increased compared to the control group, while LPE pretreatment decreased the expression levels of NOX4 and HIF2α proteins. Figure 2 (Middle D), indicating that HUA can induce mitochondrial dysfunction and amplify cellular oxidative damage, while LPE supplementation effectively alleviates oxidative stress associated with mitochondrial dysfunction.

[0094] (3) Exogenous lysophosphatidylethanolamine can alleviate liver cell mitochondrial damage and lipid peroxidation caused by high uric acid.

[0095] The mitochondrial membrane potential of cells pretreated with LPE was further detected using the JC-1 probe. The results showed that, compared with the control group (hepatocytes not treated with HUA), the mitochondrial membrane potential of hepatocytes stimulated by high concentrations of uric acid decreased, while after LPE pretreatment, the mitochondrial membrane potential significantly recovered. Figure 2 (E). Furthermore, using a kit to detect lipid peroxide levels in hepatocytes, it was found that malondialdehyde (MDA) and lipid peroxide (LPO) levels were significantly elevated in the hyperuricemia (HUA) group, but these levels significantly decreased after LPE pretreatment. Figure 2 (Middle FG). This indicates that LPE can alleviate hepatocyte oxidative stress and lipid peroxidation induced by hyperuricemia. These findings reveal a potential link between mitochondrial phospholipids (mtPE) and hyperuricemia-induced mitochondrial dysfunction and ferroptosis in human hepatocytes.

[0096] Example 2: The effect of exogenous lysophosphatidylethanolamine on improving mitochondrial dysfunction in cardiomyocytes induced by hyperuricemia.

[0097] 1. Main reagents, consumables, and main instruments used in the experiment:

[0098] Table 9. Main Reagents and Consumables for the Experiment

[0099]

[0100] Table 10. Main Experimental Instruments

[0101]

[0102] 2. Cells, cell culture, and cell model establishment:

[0103] (1) The human normal cardiomyocyte line AC16 (purchased from Shanghai Cell Bank of Chinese Academy of Sciences) was cultured in DMEM medium with 10% fetal bovine serum (FBS), 100 U / mL penicillin / 100 μg / mL streptomycin added. All cells were cultured in an incubator at 37°C with 5% CO2.

[0104] (2) Establish an in vitro HUA model. The preparation method of the uric acid solution is the same as in Example 1. After the uric acid solution is prepared, AC16 cells are cultured in a medium containing 750 μmol / L UA for 48 h to establish an in vitro HUA model.

[0105] (3) To evaluate the effect of lysophosphatidylethanolamine, an LPE treatment model was established: LPE was first dissolved in ethanol and then diluted in culture medium to prepare a final concentration of 50 μM. 50 μmol / L LPE was added to AC16 cells and 750 μmol / L UA was added at the same time to stimulate for 48 h.

[0106] 3. Cellular reactive oxygen species detection:

[0107] The DCFH-DA working solution was diluted to 10 μmol / L using serum-free culture medium: DCFH-DA = 1000:1 (v / v). Cells were seeded in six-well plates. After drug administration and modeling, the cells were washed twice with PBS, and 1 mL of working solution was added. The cells were incubated in an incubator for 30 min. After incubation, the cells were washed three times with basal culture medium, and finally 2 mL of PBS was added for sealing. The ROS level was quantitatively analyzed using a fluorescence microscope.

[0108] 4. Western blot detection of cell-related protein levels:

[0109] (1) Sample preparation: The procedure is the same as in Example 1.

[0110] (2) Determination of total protein concentration by BCA method: The procedure is the same as in Example 1.

[0111] (3) Protein denaturation: The procedure is the same as in Example 1.

[0112] (4) Preparation of SDS-PAGE gel: The procedure is the same as in Example 1.

[0113] (5) Western Blot electrophoresis: The procedure is the same as in Example 1.

[0114] (6) Western Blot transfer: The operation is the same as in Example 1.

[0115] (7) Closure: Same as in Example 1.

[0116] (8) Incubation with primary and secondary antibodies: Same as in Example 1.

[0117] (9) Development: The operation is the same as in Example 1.

[0118] 5. Detection of mitochondrial membrane potential (JC-1): AC16 cells were subjected to a 1×10⁻⁶ ion exchange rate. 5 Cells were seeded at a density of / wells in 24-well plates. After HUA or LPE treatment, 200 µL of serum-free medium containing dissolved JC-1 probe (5 µg / mL) was added, and the cells were incubated at 37°C for 20–30 min. Cells were washed twice with staining buffer (1×). After washing, 500 µL of medium was added to each well. Cell fluorescence was detected under a fluorescence microscope, and fluorescence intensity was quantified using ImageJ software.

[0119] 6. Electron microscopy: AC16 cells were seeded in 6 cm culture dishes, treated with UA (750 μM), collected, and counted. 1 × 10⁶ cells were used. 6 Cell samples were slowly added along the tube wall into 2.5% glutaraldehyde fixative and then stored at 4°C. They were then subjected to a gradient ethanol dehydration process (30%, 50%, 70%, 80%, 90%, 100%), critical point drying, and sample embedding. The final processed samples were observed under an electron microscope.

[0120] 7. Mitochondrial oxygen consumption assay: The oxygen consumption rate of AC16 cells was measured using a Seahorse XFe96 cellular energy metabolism analyzer. Cells were loaded at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 cells / well in Seahorse 96-well plates and cultured in DEMN medium containing 10% FPS for 24 h (37°C, 5% CO2). The plates were then stabilized at 37°C for 1 h in a CO2-free incubator. Cellular oxygen consumption rate was measured under four different conditions: (1) basal conditions (no other treatment); (2) treatment with the ATP synthase inhibitor oligomycin (1.5 μM); (3) treatment with the mitochondrial uncoupling agent FCCP (1 μM); and (4) treatment with the mitochondrial inhibitors rotenone and antimycin A (0.5 μM each). The data were analyzed and plotted using Seahorse Wave software.

[0121] 8. Experimental Results:

[0122] (1) High uric acid induces AC16 mitochondrial dysfunction, increased oxidative stress, and apoptosis in cardiomyocytes.

[0123] To evaluate the effects of hyperuricemia stimulation on cardiomyocytes, this study established a hyperuricemia cell model by treating AC16 cardiomyocytes with 750 μmol / L uric acid for 48 h and detecting apoptotic proteins. The results showed that compared with the control group without HUA induction treatment, the HUA group exhibited a significant decrease in Bcl-2 protein levels, while the levels of BAX and cleaved Caspase-3 proteins were significantly increased, indicating enhanced apoptosis. Figure 3 (A). Flow cytometry results of cardiomyocyte apoptosis showed that HUA stimulation significantly increased apoptosis of AC16 cardiomyocytes (A). Figure 3 (B). Furthermore, the results of experiments assessing oxidative stress and mitochondrial function showed that, compared to the control group, HUA had significantly higher levels of reactive oxygen species (ROS). Figure 3 (C), while mitochondrial membrane potential and mitochondrial oxygen consumption rate decreased significantly ( Figure 3 (D, E). Experiments showed that hyperuricemia (HUA) induced AC16 mitochondrial dysfunction in cardiomyocytes, promoted apoptosis, and exacerbated oxidative stress.

[0124] (2) Exogenous lysophosphatidylethanolamine (LPE) can improve hyperuricemia (HUA)-induced cardiomyocyte damage.

[0125] To determine the relationship between hyperuricemia (HUA)-induced cardiomyocyte apoptosis, mitochondrial dysfunction, and changes in phosphatidylethanolamine (PE) levels, this study supplemented AC16 cells with 50 μM LPE under uric acid treatment and detected apoptosis-related proteins by Western blotting. The results showed that LPE treatment significantly reversed the expression of apoptosis proteins. Figure 4 (A) and reactive oxygen species levels ( Figure 4 In AC16 cells, Bcl-2 protein levels increased, while BAX and cleaved Caspase-3 protein levels decreased, and reactive oxygen species levels also decreased. Furthermore, LPE treatment significantly improved uric acid-induced mitochondrial dysfunction: mitochondrial membrane potential was significantly restored. Figure 4 In the middle C stage, mitochondrial membrane potential decreases, and mitochondrial respiratory function is basically fully restored. Figure 4 In the middle D, mitochondrial respiratory function is improved, and to some extent, mitochondrial structural damage caused by uric acid is reversed. Figure 4 (E). Experiments showed that LPE treatment can increase the PE content in mitochondria and correct uric acid-induced mitochondrial dysfunction, oxidative stress, and apoptosis in AC16 cells.

[0126] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. Application of lysophosphatidylethanolamine in the preparation of drugs for treating hepatocellular and / or myocardial cell damage caused by hyperuricemia.

2. The application according to claim 1, characterized in that, The lysophosphatidylethanolamine treats hepatocellular damage by improving hepatocellular mitochondrial damage and hepatocellular lipid peroxidation.

3. The application according to claim 1, characterized in that, The lysophosphatidylethanolamine treats hepatocellular damage by improving hepatocyte iron overload and oxidative stress.

4. The application according to claim 1, characterized in that, The lysophosphatidylethanolamine treats cardiomyocyte damage by reducing cardiomyocyte apoptosis and improving mitochondrial damage in cardiomyocytes.

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