Use of ornithine L-aspartate in the preparation of a drug for treating systemic senescence

Abstract

This invention discloses the application of ornithine aspartate in the preparation of drugs for treating systemic aging. By applying ornithine aspartate to regulate the systemic aging process, this invention can effectively improve hepatocyte dysfunction, restore cell proliferation activity, alleviate cell cycle arrest, and reduce the abnormal expression of aging-related genes and proteins in multiple tissues and organs such as the liver, kidneys, heart, muscles, and fat, thereby delaying the aging process. This technical solution can significantly improve the structural disorder and lipid deposition in aging liver tissue, reduce serum transaminase levels, improve liver function, reduce insulin resistance, inhibit the overexpression of SASP-related inflammatory factors, and maintain metabolic homeostasis. Furthermore, it effectively improves aging phenotypes in multiple tissues and organs. Therefore, this invention has significant technical effects and application potential in delaying systemic aging and improving motor function and metabolic abnormalities.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of ornithine aspartate in the preparation of drugs for treating systemic aging. Background Technology

[0002] Systemic aging, a key issue in the biomedical field, is a complex process involving the gradual decline of function in multiple organs and systems throughout the body. With the increasing aging of the global population, systemic aging and its related diseases, such as cardiovascular diseases, neurodegenerative diseases, and metabolic diseases, have become major public health problems seriously threatening human health and quality of life. These age-related diseases not only cause immense suffering for patients but also impose a heavy economic burden on families and society. For example, Alzheimer's patients gradually lose cognitive function, become unable to care for themselves, and require long-term care; cardiovascular diseases can lead to serious consequences such as acute myocardial infarction and stroke, even endangering life. Therefore, in-depth research into the mechanisms of systemic aging and the search for effective interventions are of paramount practical significance.

[0003] Currently, interventions for age-related diseases mainly include lifestyle interventions, drug therapy, and rehabilitation therapy. 1. Lifestyle interventions such as a balanced diet, moderate exercise, and quitting smoking and limiting alcohol consumption can help slow down the aging process, but their effects are limited and difficult to maintain in the long term.

[0004] 2. Regarding drug treatment, most existing medications can only relieve symptoms and cannot fundamentally reverse the aging process, and they also have certain side effects. For example, some drugs used to treat neurodegenerative diseases may cause adverse reactions such as gastrointestinal discomfort and dizziness.

[0005] 3. Rehabilitation therapy mainly focuses on functional recovery training for the sequelae of diseases, and has little effect on preventing the occurrence of age-related diseases.

[0006] Current interventions for systemic aging have significant limitations. In drug therapy, traditional treatments primarily focus on symptom relief, failing to fundamentally slow down or even reverse the systemic aging process. Furthermore, long-term use easily leads to drug resistance and side effects, increasing the metabolic burden on the liver and kidneys. While gene therapy shows promise, the technology is still immature, carrying the risk of off-target gene editing and potentially causing uncontrollable gene mutations. Stem cell transplantation faces challenges such as limited cell sources, high culture costs, and difficulty in precisely controlling post-transplant cell survival and differentiation regulation.

[0007] Therefore, given these limitations, the search for new drugs to treat systemic aging is urgent and there is an urgent need to find new and more effective interventions to improve the condition of systemic aging and prevent and treat age-related diseases. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of ornithine aspartate in the preparation of drugs for treating systemic aging. The use of ornithine aspartate in this invention is expected to break through the existing difficulties and achieve safer, more effective and precise treatment, bringing new hope for the prevention and treatment of systemic aging-related diseases and having great significance for maintaining a high quality of life.

[0009] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides the application of ornithine aspartate in the preparation of drugs for treating systemic aging.

[0010] The present invention also provides a medicament for treating systemic aging, the medicament comprising ornithine aspartate.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the excipient is any one of physiological saline, glucose, and vitamin C.

[0013] Furthermore, the drug is an oral preparation, a nasal drop, or an injectable preparation.

[0014] Furthermore, the oral preparation is a tablet, capsule, or granule.

[0015] The principle of this invention: L-ornithine L-aspartate (LOLA), also known as L-ornithine-L-aspartate or L-ornithine-L-aspartic acid, with the chemical name (S)-2,5-diaminovaleric acid-(S)-aminosuccinate, is a naturally occurring amino acid compound that plays an important role in human metabolism. It is currently a primary clinical drug used to treat liver diseases (such as hepatic encephalopathy, acute and chronic hepatitis). Its mechanism of action includes promoting hepatocyte repair and regeneration, improving liver metabolic function, and reducing blood ammonia levels, thereby alleviating liver disease symptoms. In terms of pharmacokinetics, LOLA reaches peak plasma concentration 30-60 minutes after oral administration, with a bioavailability of approximately 82%, and its metabolites are excreted in the urine. It rapidly participates in hepatocyte metabolism, promoting ammonia conversion and excretion, and provides energy to hepatocytes by enhancing the tricarboxylic acid cycle, supporting the repair of damaged hepatocytes.

[0016] Recent studies have shown that LOLA may have the potential to intervene in aging. It can regulate metabolic pathways, enhance cellular antioxidant capacity, and reduce oxidative stress damage—processes closely related to aging mechanisms. Furthermore, LOLA can improve mitochondrial function and increase cellular energy metabolism, thereby enhancing cell vitality and function. Regarding liver aging, since liver aging is often accompanied by decreased hepatocyte function and abnormal ammonia metabolism, LOLA's effects in improving ammonia metabolism and promoting hepatocyte repair suggest potential value in delaying liver aging and improving liver function; however, direct research on this aspect is currently lacking. Although research on its application in other tissues and organs is still limited, in vitro experiments have shown that LOLA can significantly improve inflammation and oxidative stress, suggesting its potential in systemic aging intervention.

[0017] In summary, LOLA is not only a safe and effective drug for treating liver disease, but it also has the potential to become a novel drug for intervening in aging and related diseases, with potential for further research and application.

[0018] The beneficial effects of this invention are: This invention utilizes ornithine aspartate (LOLA) to regulate the systemic aging process, effectively improving hepatocyte dysfunction, restoring cell proliferation activity, alleviating cell cycle arrest, and reducing the abnormal expression of aging-related genes and proteins in multiple tissues and organs such as the liver, kidneys, heart, muscles, and fat, thereby delaying the aging process. This technical solution can significantly improve the structural disorder and lipid deposition in aging liver tissue, reduce serum transaminase levels, enhance liver function, reduce insulin resistance, inhibit the overexpression of SASP-related inflammatory factors, and maintain metabolic homeostasis. Furthermore, it effectively improves aging phenotypes in multiple tissues and organs. Therefore, this invention has significant technical effects and application potential in delaying systemic aging and improving motor function and metabolic abnormalities. Attached Figure Description

[0019] Figure 1 The effect of ornithine aspartate on the proliferation level of doxorubicin-induced senescent hepatocytes is shown in the figure. in, This means: p < 0.0001. This indicates that p < 0.05; Figure 2 Figure showing the effects of ornithine aspartate on β-galactosidase and Oil Red O staining in hepatocytes of aging mice. Figure 3 The effect of ornithine aspartate on the expression levels of cell cycle-related proteins in doxorubicin-induced senescent hepatocytes is shown in the figure. In the figure, A is the protein expression detection map, and B is the RNA expression detection map. Figure 4The effect of ornithine aspartate on the cell cycle of doxorubicin-induced senescent hepatocytes is shown in the figure. In the figure, A is a cell cycle distribution diagram, and B is a cell cycle protein expression detection diagram. Figure 5 The effect of ornithine aspartate on liver weight and liver function in aging mice is shown in the figure. In the image, A shows the mouse weight monitoring data, and B shows the liver transaminase detection data. Figure 6 Figure showing the effect of ornithine aspartate on the gene expression levels of liver inflammatory factors in aging mice; Figure 7 The effect of ornithine aspartate on glucose tolerance levels in aging mice is shown in the figure. Figure 8 Figure showing the effect of ornithine aspartate on the overall metabolic level of aging mice; In the figure, A is a metabolic heat production graph, B is an oxygen consumption monitoring graph, and C is a carbon dioxide emission monitoring graph. Figure 9 Figure showing the effect of ornithine aspartate on the motor function of aging mice; In the diagram, A is the rotator experiment diagram, and B is the track experiment diagram. Figure 10 The effect of ornithine aspartate on β-galactosidase in the liver and kidneys of aging mice is shown in the figure. Figure 11 The effect of ornithine aspartate on H&E staining of liver, kidney, heart, small intestine and spleen in aging mice; Figure 12 The effect of ornithine aspartate on the expression levels of cyclin in the liver, kidney, muscle, and adipose tissue of aging mice is shown in the figure. In the diagram, A represents liver protein testing, B represents kidney protein testing, C represents muscle protein testing, and D represents lipoprotein testing. Figure 13 The figure shows the effect of ornithine aspartate on the expression levels of cyclin in the heart, small intestine, and spleen of aging mice. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0021] Example 1: Effect of aspartate ornithine (LOLA) on DOX-induced proliferation of AML12 cells 1. Experimental Materials 1.1 Cell lines: Mouse hepatocyte line AML12. Cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin in a 37°C, 5% CO2 incubator.

[0022] 1.2 Drugs and Reagents: Doxorubicin (DOX), ornithine aspartate (LOLA), EdU cell proliferation assay kit, DAPI nuclear dye, PBS buffer, ethanol, trypsin, paraformaldehyde, Triton X-100.

[0023] 1.3 Main Instruments and Equipment: Cell culture incubator, inverted fluorescence microscope, centrifuge 2. Experimental Methods 1) AML12 cells were harvested during the logarithmic growth phase and seeded into 96-well plates, with approximately 1 × 10⁶ cells per well. 4 Cells were cultured for 24 hours to ensure stable adhesion. Subsequently, they were treated with drugs and divided into three groups: a control group, a DOX group, and a DOX+LOLA combined treatment group.

[0024] Control group: Cells were cultured normally without any drugs; DOX group: Cells were treated with doxorubicin at a final concentration of 0.5 μM for 24 h; DOX+LOLA group: LOLA with a final concentration of 5mM was added 2 hours before the addition of doxorubicin, and then it was combined with DOX for 24 hours.

[0025] 2) After drug treatment, the medium was replaced with 10 μM EdU and incubated for another 2 hours to label proliferating cells undergoing DNA synthesis. The culture medium was then discarded, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and treated with 0.5% Triton X-100 solution at room temperature for 20 min to increase cell membrane permeability. The reaction solution was prepared and incubated according to the EdU staining reaction system requirements, and the reaction was carried out in the dark for 30 min. After staining, the cells were washed with PBS and the nuclei were stained with DAPI. The red (EdU positive) and blue (nucleus) fluorescence signals were observed under a microscope.

[0026] 3) After observation, multiple fields of view were randomly selected for photography. Image analysis software was used to count the number and positivity rate of EdU-positive cells. Each experiment was repeated three times, and data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and a p < 0.05 was considered statistically significant.

[0027] 3. Experimental Results EdU fluorescence staining results showed ( Figure 1In the control group, strong EdU-positive signals indicated active cell proliferation. DOX treatment significantly reduced the number of EdU-positive cells, demonstrating that DOX significantly inhibited AML12 cell proliferation. However, in the DOX+LOLA combined treatment group, the number of EdU-positive cells significantly increased, and the fluorescence signal was enhanced compared to the DOX group, indicating that LOLA effectively mitigated the inhibitory effect of DOX on cell proliferation. These combined results suggest that LOLA can significantly improve doxorubicin-induced hepatocyte proliferation impairment, providing experimental evidence for its application in the prevention and treatment of drug-induced liver injury and liver function decline.

[0028] Example 2: Effects of LOLA on senescence phenotype and lipid accumulation in hepatocytes of aging mice 1. Experimental Materials 1.1 Laboratory animals: The experiment used male C57BL / 6 mice, including a young group (3 months old) and a naturally aged group (20 months old). All mice were housed in a temperature-controlled (22±2°C) and humidity-controlled (50–60%) animal room with a 12-hour light / dark cycle and free access to standard feed and water. The experimental procedures strictly followed the ethical guidelines for laboratory animals and were conducted after approval by the institution's animal ethics committee.

[0029] 1.2 Drugs and Reagents: The main drug used in the experiment was ornithine aspartate (LOLA).

[0030] Reagents used for the isolation and culture of primary hepatocytes include type I collagenase, Hank's balanced salt solution, DMEM medium, fetal bovine serum, penicillin and streptomycin, and trypsin.

[0031] Cellular senescence was detected using a β-galactosidase staining kit, lipid droplet detection was performed using Oil Red O staining solution, and nuclear staining was performed using hematoxylin or DAPI.

[0032] Standard laboratory-grade reagents such as PBS, formaldehyde, isopropanol, and glycerol mounting solution are commonly used.

[0033] 1.3 Instruments and Consumables: The main instruments used in the experiment included a clean bench, an incubator, an inverted microscope, a centrifuge, a water bath, and a microscopic imaging system. Routine experimental consumables included sterile petri dishes, glass coverslips, pipettes, and filter tips.

[0034] 2. Experimental Methods 2.1 Extraction and culture of primary hepatocytes Mice were anesthetized and their livers were exposed via thoracotomy. Hepatocytes were isolated using an in situ two-step perfusion method via portal vein cannulation. First, the liver was perfused with pre-warmed calcium- and magnesium-free Hank's buffer to remove blood. Then, a digestive solution containing type I collagenase was infused for tissue digestion. After removing the liver, the tissue was gently dispersed with sterile forceps and filtered. The resulting suspension was centrifuged at low speed, and the hepatocyte pellet was collected. The cells were resuspended in DMEM medium containing 10% fetal bovine serum and seeded in collagen-coated culture dishes. The cells were cultured at 37°C in a 5% CO2 incubator until they were stable and ready for further processing.

[0035] 2.2 Experimental Grouping and Processing Primary hepatocytes isolated from senescent mice were randomly divided into two groups: Control group: Young mouse hepatocytes served as physiological controls.

[0036] LOLA treatment group: LOLA was added to the culture medium at a final concentration of 5 mM and left to stand for 24 h; the control group received no drug.

[0037] 2.3 β-Galactosidase staining for cellular senescence detection Cells in each group were washed with PBS, fixed with fixative for 15 min, and then incubated overnight at 37°C in the dark without CO2 using β-galactosidase staining working solution. The cells were observed and photographed under a microscope the following day; blue staining indicated a positive senescence signal. The proportion of positive cells in random fields of view was counted to reflect the degree of senescence.

[0038] 2.4 Detection of lipid droplet accumulation by Oil Red O staining Mouse hepatocyte line AML12 cells were cultured under different conditions, washed with PBS, and fixed with 4% paraformaldehyde for 15 min. They were then rinsed with 60% isopropanol and stained with Oil Red O at room temperature for 30 min. After staining, excess dye was washed away, and the cell nuclei were counterstained with DAPI or hematoxylin. The distribution of red lipid droplets was observed under a microscope. The percentage of lipid droplet area was determined using image analysis software.

[0039] 2.5 Statistical Analysis Each experiment was repeated three times, and data are expressed as mean ± standard deviation. One-way ANOVA was used to analyze differences between groups, with a significance level set at p < 0.05.

[0040] 3. Experimental Results The results show that ( Figure 2 In senescent mouse primary hepatocytes, β-galactosidase staining was significantly deepened, and the proportion of positive cells was significantly higher than that in the younger group. Oil Red O staining of senescent AML12 cells showed a large amount of lipid droplet deposition, indicating significant metabolic abnormalities in senescent cells. After LOLA treatment, β-galactosidase staining was significantly reduced, lipid droplet accumulation decreased, and cell morphology tended to return to normal.

[0041] The above results indicate that LOLA can effectively reduce the senescent phenotype and lipid deposition in hepatocytes of aging mice, and has the effect of improving hepatocyte metabolic function and delaying cell senescence.

[0042] Example 3: Effects of LOLA on DOX-induced expression of cell cycle-related proteins and genes in AML12 cells. 1. Experimental Materials 1.1 Cells: The cells used in the experiments were the mouse hepatocyte line AML12, cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, and maintained stable growth at 37°C and 5% CO2. All experiments were performed by treating cells during the logarithmic growth phase.

[0043] 1.2 Drugs and Reagents: The drugs used in the experiment included doxorubicin (DOX) and ornithine aspartate (LOLA). DOX was used to induce cell cycle arrest and upregulation of senescence-related molecules, while LOLA was used to detect its reversal effect on this process.

[0044] Other reagents include protein lysis buffer, BCA protein quantification reagent, SDS-PAGE gel, electroporation buffer, blocking solution, primary and secondary antibodies, ECL imaging reagent, RNA extraction reagent, reverse transcription kit, and real-time quantitative PCR related reagents.

[0045] 1.3 Instruments and Consumables: The main instruments used in the experiment included a constant temperature cell culture incubator, an inverted microscope, a benchtop centrifuge, an electrophoresis and transfer system, a constant temperature PCR instrument, and a real-time fluorescence quantitative PCR instrument.

[0046] Standard laboratory consumables include sterile petri dishes, EP tubes, pipette tips, centrifuge tubes, glass slides, and anti-contamination filter tips.

[0047] 2. Experimental Procedure 1) AML12 cells were seeded in the logarithmic growth phase into 6-well plates, approximately 2 × 10⁶ cells per well. 5 Cells were cultured for 24 hours and then treated with the drug. The experiment was divided into a control group, a DOX group, and a DOX+LOLA combined treatment group.

[0048] Control group: No medication added. DOX group: Doxorubicin at a final concentration of 0.5 μM was added and the treatment lasted for 24 hours. DOX+LOLA group: LOLA at a final concentration of 5mM was added 2 hours before the addition of doxorubicin and the mixture was incubated together for 24 hours.

[0049] 2) After drug treatment, protein and RNA extraction were performed separately. Protein samples were lysed, centrifuged, and the supernatant was quantified using BCA. An equal volume of protein was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder for 1 hour, followed by the addition of primary antibodies against p16, p21, p53, and β-actin, and incubated overnight at 4°C. The next day, the corresponding HRP-labeled secondary antibodies were added, and the membrane was incubated at room temperature for 1 hour. The membrane was then developed using ECL chemiluminescence immunoassay, and the signal was recorded. β-actin was used as an internal control for normalized analysis.

[0050] 3) Statistical analysis: Total RNA was extracted from cells in each group, and cDNA was obtained by reverse transcription. The transcriptional levels of p16, p21, and p53 were detected using real-time quantitative PCR. The PCR reaction system was prepared according to the manufacturer's instructions, and the cycling conditions were 95°C pre-denaturation, 60°C annealing, and 72°C extension, for a total of 40 cycles. Relative expression levels were calculated using the 2^-ΔΔCt method, with GAPDH as an internal control. Each experiment was repeated three times, and the results are expressed as mean ± standard deviation. Data were analyzed using one-way ANOVA, with p < 0.05 being the criterion for statistical significance.

[0051] 3. Experimental Results WB and RT-qPCR results showed that ( Figure 3 DOX treatment significantly increased the protein and transcriptional levels of p16, p21, and p53 in AML12 cells, suggesting that cell cycle arrest and senescence-related pathways were activated. Compared with the DOX group, the expression of these molecules was significantly reduced in the DOX+LOLA combined treatment group, indicating that LOLA can effectively inhibit DOX-induced upregulation of cyclins and alleviate cell cycle inhibition and senescence phenotypes.

[0052] The combined results indicate that LOLA can reverse the abnormal increases in p16, p21, and p53 induced by doxorubicin in AML12 cells, and has the potential to improve cell cycle disorder and resist cell senescence.

[0053] Example 4: Regulation of cell cycle arrest and cell cycle-related protein expression in DOX-induced AML12 cells by LOLA 1. Materials 1.1 Cells: The mouse hepatocyte line AML12 was used in the experiment, and the culture conditions were the same as those in the previous examples. All experiments used cells in the logarithmic growth phase for treatment.

[0054] 1.2 Drugs and Reagents: The drug used in the experiment was ornithine aspartate (LOLA). Cell cycle detection was performed using propidium iodide (PI) staining. Reagents used for cell lysis and protein detection included protein lysis buffer, BCA quantitative reagent, SDS-PAGE electrophoresis and transfer reagent, primary and secondary antibodies, and ECL imaging reagent. The antibodies detected included Cyclin D1 and β-actin.

[0055] 1.3 Instruments and Consumables: The main instruments include a constant temperature incubator, an inverted microscope, a flow cytometer, an electrophoresis and transfer system, an enzyme-linked immunosorbent assay (ELISA) reader, and a microscopic imaging system.

[0056] Commonly used consumables include cell culture plates, centrifuge tubes, EP tubes, filter tips, and pipettes.

[0057] 2. Experimental Procedure 1) AML12 cells were seeded in 6-well plates, approximately 2 × 10⁶ cells per well. 5 Cells were cultured for 24 hours and then divided into three groups: a control group, a DOX group, and a DOX+LOLA combined treatment group.

[0058] DOX group: Doxorubicin at a final concentration of 0.5 μM was added and the cells were treated for 24 h to induce cell cycle arrest; The DOX+LOLA group was given LOLA at a final concentration of 5 mM 2 hours before the addition of DOX, and the two groups worked together for 24 hours. The control group received no medication.

[0059] 2) Cell cycle detection: After treatment, cells were collected, washed with PBS, and fixed overnight with 70% ethanol. After fixation, cells were washed with PBS, and PI staining solution containing RNase A was added. Cells were incubated at 37°C in the dark for 30 min. Cell DNA content was detected by flow cytometry, and the proportions of cells in G0 / G1, S, and G2 / M phases were analyzed using FlowJo software to assess cell cycle distribution.

[0060] 3) Protein Expression Detection: Cells from each group were collected simultaneously for protein analysis. After cell lysis and centrifugation, the supernatant was collected for BCA quantification. An equal volume of protein sample was used for SDS-PAGE separation and transfer to a membrane. The membrane was blocked with 5% skim milk powder for 1 hour, and primary antibodies against Cyclin D1 and β-actin were added, respectively, and incubated overnight at 4°C. The next day, the corresponding HRP-labeled secondary antibodies were added, and the membrane was incubated at room temperature for 1 hour before signal detection using ECL imaging. β-actin was used as an internal control for quantification.

[0061] 4) Statistical analysis: All experiments were repeated three times, and results are expressed as mean ± standard deviation. Data were compared using one-way ANOVA, with a significance level set at p < 0.05.

[0062] 4. Experimental Results Flow cytometry results showed ( Figure 4 A): DOX treatment significantly increased the proportion of AML12 cells in S phase, indicating that the cell cycle was arrested in the S phase; however, after LOLA treatment, the proportion of S phase cells decreased significantly and the proportion of G1 phase cells recovered, indicating that LOLA can effectively reverse DOX-induced cell cycle arrest.

[0063] WB test results show ( Figure 4 B): DOX treatment led to a decrease in Cyclin D1 protein levels; while LOLA combined treatment significantly restored Cyclin D1 expression.

[0064] The combined results indicate that LOLA can reverse DOX-induced cell cycle arrest in AML12 cells, restore normal cell cycle distribution, and maintain cell proliferation activity by regulating the expression of Cyclin D1 and cell cycle inhibitory proteins, suggesting its potential role in improving hepatocyte aging-related cell cycle disorders.

[0065] Example 5: The effect of LOLA on improving liver function and insulin resistance in aging mice. 1. Materials 1.1 Laboratory animals: The experiment used male C57BL / 6 mice, including a young group (3 months old) and a naturally aged group (20 months old). Mice were housed under constant temperature and humidity conditions with a 12-hour light-dark cycle and free access to standard feed and water. All experiments were approved by the animal ethics committee and conducted in accordance with relevant regulations.

[0066] 1.2 Drugs and Reagents: The drug used in the experiment was ornithine aspartate (LOLA), which was administered via drinking water.

[0067] PBS, physiological saline, isopropanol and ethanol used in routine experiments are analytical grade reagents.

[0068] 1.3 Instruments and Consumables: The main instruments include an automated biochemical analyzer, a micro-ELISA reader, a centrifuge, and a blood glucose meter.

[0069] Commonly used consumables include disposable syringes, blood collection needles, centrifuge tubes, and sterile feeding equipment.

[0070] 2. Experimental Procedure 1) The experimental mice were divided into three groups: young control group, aged control group and LOLA treatment group.

[0071] LOLA treatment group: Mice were administered the drug continuously via drinking water at a final concentration of 2 g / kg / day for 8 weeks; Younger and older groups: Provided with regular drinking water.

[0072] During the experiment, all mice in each group were allowed free access to food and water, and their weight and water intake were monitored regularly to ensure consistency in drug administration.

[0073] 2) After the drug administration period, mice were fasted for 12 hours and then anesthetized for blood collection. Serum ALT and AST activities were measured using an automated biochemical analyzer to assess changes in liver function. A glucose tolerance test (GTT) was then performed to assess hepatic glucose metabolism. Mice were injected with glucose solution (2 g glucose / g body weight), and tail vein blood glucose was measured at 0, 30, 60, 90, and 120 minutes. The area under the blood glucose curve (AUC) was calculated to reflect glucose metabolism efficiency.

[0074] 3) Statistical analysis: All experiments were repeated three times, and results are expressed as mean ± standard deviation. Data were compared using one-way ANOVA, with a significance level set at p < 0.05.

[0075] 4. Experimental Results The results showed that serum ALT and AST levels in the aging control group were significantly higher than those in the younger group, indicating liver function impairment. Figure 5 B); at the same time, its AUC under the GTT curve decreased significantly, indicating a significant reduction in glucose metabolism (B). Figure 7 After 8 weeks of LOLA drinking water intervention, the serum ALT and AST levels in mice were significantly reduced. Figure 5 B), glucose tolerance was significantly improved ( Figure 7 The above results indicate that LOLA can effectively improve liver function damage and alleviate insulin resistance in aging mice, suggesting its potential application value in delaying age-related metabolic abnormalities.

[0076] Example 6: Effect of LOLA on SASP gene expression in liver tissue of aging mice 1. Materials 1.1 Laboratory animals: The experiment used male C57BL / 6 mice, including a young group (3 months old) and a naturally aged group (20 months old). Animal housing conditions were the same as in the previous examples: a constant temperature and humidity environment, a 12-hour light / dark cycle, and free access to food and water. All experimental procedures were approved by the animal ethics committee and conducted in accordance with animal experimental standards.

[0077] 1.2 Drugs and Reagents: The drug used in the experiment was ornithine aspartate (LOLA), which was administered via drinking water.

[0078] The reagents used for RNA extraction include RNA extraction solution, reverse transcription kit, and real-time quantitative PCR detection reagent.

[0079] The primers used for detecting the main genes in the experiment included SASP-related molecules: IL-1α, IL-1β, IL-6, TNF-α, and CCL2, with β-actin as the internal reference gene. Commonly used buffers, RNase-free water, and other molecular biology grade reagents were prepared and used according to standard procedures.

[0080] 1.3 Instruments and Consumables: The main instruments include a real-time quantitative PCR instrument, a constant temperature centrifuge, and a clean bench.

[0081] Commonly used consumables include enzyme-free RNA centrifuge tubes, pipettes, and matching pipette tips.

[0082] 2. Experimental Procedure 1) The experiment was divided into a young control group, an aging control group, and a LOLA treatment group.

[0083] LOLA mice were administered the drug continuously via drinking water at a final concentration of 2 g / kg / day for 8 weeks. The younger group and the aging control group drank ordinary drinking water.

[0084] After the experiment, the mice were anesthetized and euthanized, and their liver tissue was quickly taken, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0085] 2) Approximately 50 mg of liver tissue was collected, and total RNA was extracted using RNA extraction buffer. Purity and concentration were determined by UV spectrophotometry. An equal volume of RNA was reverse transcribed into cDNA, and the mRNA expression level of SASP-related genes was detected using real-time quantitative PCR. The PCR reaction system and cycling conditions were performed according to the kit instructions, with the reaction program set to 95°C pre-denaturation, 60°C annealing, and 72°C extension, for a total of 40 cycles. Relative expression levels were calculated using the 2^-ΔΔCt method, with β-actin as an internal control.

[0086] 3) Statistical analysis: All experiments were repeated three times, and results are expressed as mean ± standard deviation. Data were compared using one-way ANOVA, with a significance level set at p < 0.05.

[0087] 4. Experimental Results RT-qPCR results showed that ( Figure 6In aged mice, the mRNA levels of SASP-related genes such as IL-1α, IL-1β, IL-6, TNF-α, and CCL2 in liver tissue were significantly higher than those in the young group, suggesting the activation of inflammation-related secretory phenotypes in aged liver tissue. After 8 weeks of LOLA intervention, SASP gene expression levels were significantly reduced, approaching the levels of the young group. These results indicate that LOLA can effectively reduce inflammatory SASP gene expression in the liver tissue of aged mice, suggesting its potential role in improving the liver inflammatory microenvironment and delaying tissue aging.

[0088] Example 7: Effects of LOLA on the morphology and aging phenotype of various tissues in aging mice 1. Materials 1.1 Laboratory animals: The experiment used male C57BL / 6 mice, including a young group (3 months old) and a naturally aged group (20 months old). Animal housing conditions were the same as in the previous examples: a constant temperature and humidity environment, a 12-hour light / dark cycle, and free access to food and water. All experimental procedures were approved by the animal ethics committee and conducted in accordance with animal experimental standards.

[0089] 1.2 Drugs and Reagents: The experimental drug was ornithine aspartate (LOLA), administered via drinking water.

[0090] The reagents used for histological testing include 4% paraformaldehyde fixative, paraffin embedding material, hematoxylin-eosin (HE) staining solution, β-galactosidase staining reagent, Oil Red O staining solution, and glycerol gelatin mounting medium.

[0091] PBS, isopropanol, and physiological saline required for routine experiments are all analytical grade reagents.

[0092] 1.3 Instruments and Consumables: The main instruments include a tissue microtome, an optical microscope, a cryostat, and a constant temperature oven. Commonly used consumables include glass slides, coverslips, filter paper, and mounting materials.

[0093] 2. Experimental Procedure 1) The experiment was divided into a young control group, an aging control group, and a LOLA treatment group.

[0094] LOLA group: Mice were administered the drug continuously via drinking water, with a final concentration of 2 g / kg / day, for 8 weeks; Young group and aging control group: drank ordinary drinking water.

[0095] After the experiment, the mice were anesthetized and euthanized. Parts of each tissue were used for paraffin embedding, and the remaining tissues were rapidly frozen for later use.

[0096] 2) HE staining: Fixed tissue was routinely dehydrated, embedded in paraffin, and sectioned (approximately 5 μm thick). After dewaxing and rehydration, the sections were sequentially stained with hematoxylin, differentiated, blued, and counterstained with eosin. After dehydration and clearing, the sections were mounted. Cell morphology and histological changes were observed under a microscope.

[0097] 3) β-Galactosidase staining: Fresh tissue sections were washed with PBS and incubated with β-galactosidase staining solution (pH 6.0) at 37°C for 12–16 h. After staining, the sections were washed with PBS and mounted. The blue-green positive signal was observed under a microscope, reflecting the degree of cell senescence.

[0098] 4) Statistical analysis: Five fields of view were randomly selected from all slices for image acquisition and semi-quantitative analysis. Results are expressed as mean ± standard deviation. Data were compared using one-way ANOVA, with a significance level set at p < 0.05.

[0099] 4. Experimental Results β-galactosidase staining results showed that ( Figure 10 In the aging group, the expression of β-galactosidase in liver and kidney cells was significantly increased, indicating obvious aging characteristics, while the expression of β-galactosidase was significantly reduced in the LOLA-treated group. HE staining of mouse liver, kidney, heart, small intestine, and spleen showed... Figure 11 In the aging group, various tissues exhibited signs of aging, including cell atrophy, disordered cell arrangement, and structural disorder. Some cells showed vacuolar degeneration and inflammatory cell infiltration. After LOLA treatment, the tissue structure of mice was significantly improved, cell morphology returned to normal, and inflammatory manifestations were reduced. The overall results indicate that LOLA can significantly improve the disordered cell morphology and structure of aging mouse tissues and has the effect of reducing tissue pathological damage.

[0100] Example 8: Effects of LOLA on cyclin expression in various tissues of aging mice 1. Materials 1.1 Laboratory animals: The experiment used male C57BL / 6 mice, including a young group (3 months old) and a naturally aged group (20 months old). Animal housing conditions were the same as in the previous examples: a constant temperature and humidity environment, a 12-hour light / dark cycle, and free access to food and water. All experimental procedures were approved by the animal ethics committee and conducted in accordance with animal experimental standards.

[0101] 1.2 Drugs and Reagents: The experimental drug was ornithine aspartate (LOLA), administered via drinking water.

[0102] The main reagents used in immunofluorescence staining include 4% paraformaldehyde fixative, blocking solution, anti-p21 antibody, fluorescently labeled secondary antibody, DAPI staining solution, and anti-fluorescence quenching mounting medium.

[0103] Commonly used reagents such as PBS, physiological saline, and glycerol buffer are all analytical grade reagents.

[0104] 1.3 Instruments and Consumables: The main experimental equipment includes a fluorescence microscope, a constant temperature incubator, a centrifuge, and a microtome. Commonly used consumables include glass slides, coverslips, pipette tips, and centrifuge tubes.

[0105] 2. Experimental Procedure 1) The experiment was divided into a young control group, an aging control group, and a LOLA treatment group.

[0106] LOLA mice were administered the drug continuously via drinking water at a final concentration of 2 g / kg / day for 8 weeks. The younger group and the aging control group drank ordinary drinking water.

[0107] After the experiment, the mice were anesthetized and euthanized. Heart, small intestine and spleen tissues were quickly taken and fixed with 4% paraformaldehyde for 12 hours before routine dehydration, paraffin embedding and sectioning (thickness about 5 μm).

[0108] 2) Protein Expression Detection: Tissue samples were collected simultaneously for protein analysis. After lysis and centrifugation, the supernatant was used for BCA quantification. Equal volumes of protein samples were separated by SDS-PAGE and transferred to a membrane. The membrane was blocked with 5% skim milk powder for 1 hour, and primary antibodies against P21, P53, and β-actin were added, followed by incubation at 4°C overnight. The next day, the corresponding HRP-labeled secondary antibodies were added, and the membrane was incubated at room temperature for 1 hour before signal detection using ECL imaging. β-actin was used as an internal control for quantification.

[0109] 3) Immunofluorescence staining: After dewaxing and rehydration, tissue sections were heat-retarded with citrate buffer, washed with PBS, and blocked with blocking buffer containing 5% serum at room temperature for 1 hour. Anti-p21 primary antibody was then added, and the sections were incubated overnight at 4°C. The following day, after washing, fluorescently labeled secondary antibody was added, and the sections were incubated at room temperature for 1 hour in the dark. Finally, the cell nuclei were counterstained with DAPI, and the sections were mounted with anti-fluorescence quenching mounting medium. The sections were observed and photographed under a fluorescence microscope.

[0110] 4) Statistical analysis: Five fields of view were randomly selected from all slices for image acquisition and semi-quantitative analysis. Results are expressed as mean ± standard deviation. Data were compared using one-way ANOVA, with a significance level set at p < 0.05.

[0111] 4. Experimental Results Protein test results showed ( Figure 12In aging control mice, the expression of P21 and P53 in the liver, kidney, muscle, and adipose tissue was significantly increased compared to that in young mice, indicating tissue aging. In the LOLA-treated group, the expression of P21 and P53 in the corresponding tissues was significantly decreased. Immunofluorescence results showed ( Figure 13 In the aging control group mice, p21 positive signals were significantly enhanced and fluorescence intensity was high in the heart, small intestine, and spleen tissues, indicating overexpression of cell cycle repressor proteins and their association with tissue aging. After LOLA treatment, p21 fluorescence signals in all tissues were significantly weakened, and the proportion of positive cells decreased, indicating that LOLA can effectively inhibit the abnormal increase of p21 in aging tissues. These results suggest that LOLA can alleviate the abnormal expression of cell cycle repressor proteins and improve tissue cell activity at multiple tissue levels, indicating its potential for systemic anti-aging effects and maintenance of multi-organ functional homeostasis.

[0112] Example 9: Effects of LOLA on metabolism and motor function in aging mice 1. Materials 1.1 Laboratory animals: Sixteen C57BL / 6 male naturally aged mice, approximately 20 months old, were used in the experiment.

[0113] The animals were randomly divided into two groups: an aging control group and a LOLA treatment group, with 8 animals in each group.

[0114] Mice were housed under constant temperature and humidity conditions with a 12-hour light-dark cycle and free access to standard feed and water. All experiments were approved by the animal ethics committee and conducted in accordance with relevant regulations.

[0115] 1.2 Drugs and Reagents: The drug used in the experiment was ornithine aspartate (LOLA), which was administered via drinking water.

[0116] PBS, physiological saline, isopropanol and ethanol used in routine experiments are analytical grade reagents.

[0117] 1.3 Instruments and Consumables: The main experimental equipment includes a metabolic cage system, a rotarod apparatus, and an animal treadmill. Auxiliary instruments include an electronic balance, a timing device, and a video recording system.

[0118] Commonly used consumables include sterile water bottles, blood collection tubes, disposable gloves, and feeding cages.

[0119] 2. Experimental Procedure 1) The experimental mice were divided into two groups: the aging control group and the LOLA treatment group.

[0120] LOLA treatment group: Mice were administered the drug continuously via drinking water at a final concentration of 2 g / kg / day for 8 weeks; Both the younger and older groups were given regular drinking water.

[0121] During the experiment, all groups of mice had free access to food and water, and their weight and water intake were monitored regularly to ensure consistency in drug administration. After the 8-week drug administration period, metabolic cage tests, rotarod tests, and treadmill tests were performed.

[0122] 2) Metabolic cage experiment: Each mouse was placed in a metabolic cage system for continuous monitoring for 24 hours. Real-time recording of indicators such as oxygen consumption (VO2), carbon dioxide emissions (VCO2), respiratory quotient (RQ), and energy consumption was conducted to assess basal metabolic level and metabolic efficiency.

[0123] 3) Spinning bar test: A spinning bar device was used to test motor coordination. The rotation speed was set to 5 rpm at the beginning of the experiment, and increased by 1 rpm every 30 seconds until the mouse fell off the spinning bar. The duration of each mouse's fall was recorded, and the test was repeated three times and the average value was taken.

[0124] 4) Treadmill test: Used to assess exercise endurance. The initial speed of the treadmill is 10 m / min, and it is increased by 2 m / min every 5 minutes until the mouse can no longer run. The total running time and distance of the mouse are recorded.

[0125] 5) Statistical analysis: All experiments were repeated three times, and results are expressed as mean ± standard deviation. Data were compared using one-way ANOVA, with a significance level set at p < 0.05.

[0126] 4. Experimental Results The results of metabolic cage monitoring showed that ( Figure 8 The oxygen consumption, carbon dioxide emissions, and energy consumption levels of mice in the LOLA treatment group were all lower than those in the aging control group, suggesting that LOLA can reduce the overall metabolic load and improve energy utilization efficiency in aging mice.

[0127] Experimental results are as follows Figure 9 The results showed that the LOLA-treated group mice had a significantly longer dwell time on the rotarod and significantly improved motor coordination compared to the aging control group. Figure 9 A). The LOLA-treated group mice had significantly longer running time and total distance than the aging control group, demonstrating stronger exercise endurance and physical fitness. Figure 9 B).

[0128] Comprehensive analysis shows that LOLA can effectively reduce metabolic consumption in aging mice, improve their motor coordination and endurance, improve the overall metabolic state and motor function of the body, and has a positive physiological effect on delaying the aging process.

[0129] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of ornithine aspartate in the preparation of drugs for treating systemic aging.

2. A drug for treating systemic aging, characterized in that: The drug includes ornithine aspartate.

3. The drug according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

4. The drug according to claim 3, characterized in that: The excipients are any one of physiological saline, glucose, and vitamin C.

5. The drug according to claim 4, characterized in that: The drug is an oral preparation, nasal drop, or injectable preparation.

6. The drug according to claim 5, characterized in that: The oral preparation is a tablet, capsule, or granule.

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