Use of quinoline-4-carboxylic acid in the preparation of anti-aging products and products for protecting and promoting liver regeneration
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGZHOU RIJU TRADE DEVELOPMENT CO LTD
- Filing Date
- 2023-11-14
- Publication Date
- 2026-07-03
Smart Images
Figure CN117379425B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of quinoline-4-carboxylic acid in the preparation of anti-aging products and products that protect the liver and promote liver regeneration. Background Technology
[0002] The progressive loss of physiological integrity characteristic of aging leads to functional impairment and increased risk of death. For decades, biologists have faced the challenge of slowing down aging and extending lifespan. Most hibernating animals live longer than their non-hibernating counterparts, demonstrating excellent metabolic adaptation and damage protection in extreme environments. Therefore, hibernating animal models are excellent for studying metabolism, hypoxia / reperfusion, and longevity. The thirteen-striped hamster, as a hibernating animal, can tolerate low temperatures and adapt to cold by reducing the cold sensitivity of its peripheral somatosensory system and preventing cell damage caused by reactive oxygen species. Similarly, induced pluripotent stem cells (GS iPSCs) from the thirteen-striped hamster also exhibit stronger adaptability to stressful environments. Using the GS iPSC cell model, this study investigates the cold adaptation protection mechanisms of hibernating animals from a metabolic perspective, aiming to apply them to the field of anti-aging to address age-related diseases in humans.
[0003] Quinoline-4-carboxylic acid (QCA) is a quinoline monocarboxylic acid discovered through cryo-thaw metabolomics analysis of GS iPSC cells. Current data indicate that QCA belongs to the quinoline derivative class and may be a potential downstream product of tryptophan metabolism. Quinoline derivatives have attracted considerable attention due to their broad-spectrum antimalarial, anticancer, antibacterial, and antifungal effects. Furthermore, many studies suggest that quinoline derivatives have a protective effect against oxidative stress, reducing age-related oxidative damage in rats. However, due to limited existing data, it remains unclear whether QCA plays a protective role in aging and regeneration models.
[0004] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell derived from the mesoderm, possessing multipotent differentiation potential and forming an important part of the stem cell family. Currently, bone marrow-derived and umbilical cord-derived MSCs are the most studied, as they possess multipotent differentiation capabilities and the ability to secrete various cytokines, leading to their widespread clinical applications, primarily in the treatment of nervous system injuries, liver diseases, myocardial ischemia, diabetes, and skin problems. MSCs are the most widely used stem cell products on the market. With ongoing research, scientists have discovered that in addition to their multipotent differentiation potential, MSCs can also promote stem cell implantation, hematopoietic support, immune regulation, and self-replication, gradually becoming a research hotspot in this field. Since the number of cells isolated from donors is usually limited, in vitro expansion and culture of MSCs is unavoidable to obtain sufficient cells for clinical treatment. The senescence-prone nature of long-term in vitro cultured MSCs restricts their clinical development and application. Therefore, exploring the main mechanisms of MSC senescence and how to alleviate MSC senescence, as well as developing safe and effective in vitro culture anti-aging additives, are of great significance.
[0005] As the center of metabolism in the human body, the liver plays a vital role in various physiological and pathological processes, and many diseases also affect the liver. Partial hepatectomy is currently the most common and effective treatment for liver tumors, and the strong regenerative capacity of normal liver tissue is the basis for the effective implementation of hepatectomy. However, due to limitations in early diagnosis technology for liver tumors, most liver tumors are already in the middle or late stages at the time of initial diagnosis. Because of the large size of the tumor and the involvement of multiple liver segments, the volume of the residual liver after resection is insufficient to meet the body's metabolic needs, leading to life-threatening complications such as liver failure. To address the problem of insufficient residual liver volume, various surgical methods to stimulate rapid regeneration of the residual liver have been developed clinically, including portal vein ligation (PVL) / portal vein embolization (PVE) and two-stage hepatectomy combining liver partition and portal vein ligation (ALPPS). However, the efficacy of these surgeries remains controversial, and the risk of liver failure due to insufficient residual liver volume after surgery remains high. Therefore, developing safe and effective drugs to promote liver regeneration, accelerate the regeneration of residual liver in the perioperative period, and reduce the incidence of liver failure is of great significance for the surgical treatment of liver cancer.
[0006] Aging is accompanied by a significant increase in various diseases, and how to delay aging has always been a hot topic of scientific research. Stem cells play an important role in the aging process, and the aging of stem cells accelerates human aging. Mesenchymal stem cells (MSCs), as stem cells with self-renewal capacity and multi-lineage differentiation potential, have not yet achieved satisfactory results in delaying cell aging despite existing in vitro expansion culture media that allow them to be expanded and cultured for several generations. Furthermore, as mentioned above, the effectiveness of existing surgical methods to stimulate rapid regeneration of residual liver remains controversial, and the risk of liver failure in postoperative patients due to insufficient residual liver volume remains high. Therefore, further research and development of novel technologies or drugs to delay aging (stem cell aging) and promote liver regeneration after hepatectomy are needed to lay the foundation for clinical application. Summary of the Invention
[0007] To address the aforementioned technical problems, this invention provides the application of quinoline-4-carboxylic acid in the preparation of anti-aging products and products that protect the liver and promote liver regeneration. Specifically, quinoline-4-carboxylic acid can be used as a novel additive for the in vitro expansion and culture of mesenchymal stem cells, overcoming the bottleneck of easy aging of in vitro cultured mesenchymal stem cells and optimizing their clinical development and application; quinoline-4-carboxylic acid can alleviate aging-related phenotypes in fruit flies and mice, enhance the locomotion ability of fruit flies and the muscle strength of mice, and prolong the lifespan of fruit flies and mice, and is expected to become an effective ingredient in anti-aging therapeutic drugs; quinoline-4-carboxylic acid can improve the survival rate of mice after hepatectomy and promote liver regeneration after hepatectomy injury, and is expected to become an effective ingredient in drugs that promote the prognosis of patients after hepatectomy, promote liver regeneration, and alleviate or treat liver injury.
[0008] To achieve the above objectives, the present invention adopts the following technical solution:
[0009] One of the objectives of this invention is to provide the application of quinoline-4-carboxylic acid in the preparation of anti-aging products.
[0010] Furthermore, the anti-aging product specifically refers to a product that delays the aging of mesenchymal stem cells.
[0011] Furthermore, the product for delaying mesenchymal stem cell aging is specifically a product that promotes the proliferation of mesenchymal stem cells and / or a product that reduces damage to the nuclear DNA of mesenchymal stem cells.
[0012] Furthermore, the anti-aging product specifically refers to a product that enhances athletic ability and muscle strength.
[0013] The second objective of this invention is to provide the application of quinoline-4-carboxylic acid in the preparation of liver-protective products.
[0014] The third objective of this invention is to provide the application of quinoline-4-carboxylic acid in the preparation of products that promote liver regeneration.
[0015] The fourth objective of this invention is to provide the application of quinoline-4-carboxylic acid in the preparation of products that improve post-hepatectomy survival rates.
[0016] Furthermore, the liver-protecting product, the liver-regenerating product, and the product that improves post-hepatectomy survival rate are specifically products that reduce liver inflammation and / or reduce liver damage.
[0017] Compared with the prior art, the present invention has the following technical effects:
[0018] Using hibernating animals as a natural model, the inventors innovatively utilized induced pluripotent stem cells from the thirteen-striped ground squirrel to explore the molecular mechanisms and metabolic regulation mechanisms during cold adaptation and rewarming in hibernating animals. They discovered that quinoline-4-carboxylic acid (KCA) levels rise during cold adaptation and decrease during rewarming, suggesting it may be a metabolite that can mitigate stress damage. This holds promise for applications in combating age-related damage, delaying aging, and promoting regeneration and repair. Specific experiments revealed that adding KCA to the culture medium of mesenchymal stem cells in vitro can promote the proliferation of senescent mesenchymal stem cells, reduce nuclear DNA breaks, and slow down their aging; KCA can alleviate symptoms such as poor coat condition and dermatitis in aged mice; KCA can downregulate enzyme indicators of chronic inflammation and liver damage; KCA can enhance arm strength and prolong lifespan in aged mice; KCA can enhance the locomotion ability and prolong the lifespan of fruit flies; and KCA can promote liver regeneration after hepatectomy in aged mice, improve the survival rate after hepatectomy, and reduce liver damage enzyme indicators, etc. Attached Figure Description
[0019] Figure 1 This describes the DNA breakage in the nuclear cells of frozen-thawed GS iPSCs and hMSCs in Example 1 of this invention.
[0020] Figure 2 The images show volcano diagrams and violin diagrams of quinoline-4-carboxylic acid in the cryo-rewarm metabolome of GS iPSC cells in Example 1 of this invention.
[0021] Figure 3 This is a violin diagram showing the changes in the content of quinoline-4-carboxylic acid in the cold-preserved-room-temperature perfusion metabolome of hamster liver in Example 1 of the present invention;
[0022] Figure 4 This is the result of the effect of adding different concentrations of quinoline-4-carboxylic acid to the culture medium of high-generation mesenchymal stem cells on cell proliferation in Example 1 of the present invention;
[0023] Figure 5 This describes the nuclear DNA breakage and reduction of senescent cells in high-generation mesenchymal stem cells after treatment with quinoline-4-carboxylic acid in Example 1 of this invention.
[0024] Figure 6 The results of the Drosophila survival curve and tube crawling experiment after adding quinoline-4-carboxylic acid in Example 1 of this invention are shown.
[0025] Figure 7 This invention describes the changes in fur, dermatitis, arm strength, chronic inflammation, and liver damage enzyme indicators in aged mice after the addition of quinoline-4-carboxylic acid in Example 1 of the present invention.
[0026] Figure 8 This invention describes the effects of adding quinoline-4-carboxylic acid in Example 1 on the ratio of liver regeneration weight to body weight, post-hepatectomy survival rate, and liver injury enzyme indicators in mice. Detailed Implementation
[0027] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The equipment and raw materials used in the following embodiments are commercially available, and the methods used in the embodiments, unless otherwise specified, are consistent with conventionally used methods.
[0028] The technical solution of the present invention will be further described in detail below with reference to the embodiments.
[0029] Example 1
[0030] (1) GS iPSCs or hMSCs cultured under normal conditions were used as the 37℃ control group. For cold exposure treatment, the culture medium was replaced with UW medium, and after being placed at room temperature for 10 min, the cells with UW medium were transferred to a 4℃ refrigerator for storage for 48 h. For warming treatment, the cells were removed from the refrigerator and replaced with pre-cooled culture medium. After being placed at room temperature for 10 min, the cells were transferred to a 37℃ incubator for 2 h, and the samples were collected at 4℃ for 4 h and at 37℃ for 2 h. After fixing the cells with 4% paraformaldehyde, TUNEL staining was performed, and the staining results are shown in the figure. Figure 1 As shown.
[0031] The results showed that after freezing and thawing, TUNEL staining revealed an increase in the number of nuclear DNA fragments in hMSCs compared to GS iPSCs, suggesting that GS iPSCs have a stronger ability to adapt to stress environments.
[0032] (2) GS iPSCs cultured under normal conditions were used as the 37℃ control group sample. For cold exposure treatment, the GSiPSC medium was replaced with Hibernate-A medium. After being placed at room temperature for 10 min, the cells in Hibernate-A medium were transferred to a 4℃ refrigerator for 4 h, and the 4℃ 4h samples were collected. For warming treatment, the cells were removed from the refrigerator and replaced with pre-cooled GS iPSC medium. After being placed at room temperature for 10 min, the cells were transferred to a 37℃ incubator for 2 h, and the 4℃ 4h and 37℃ 2h samples were collected. After the samples were collected, they were sent to the company for metabolomics analysis. By analyzing the metabolomics data, volcano plots and violin plots were generated using a bioinformatics mapping website. The results are as follows: Figure 2 As shown.
[0033] Both the volcano plot and the violin plot show that quinoline-4-carboxylic acid increases during cold adaptation and decreases during rewarming.
[0034] (3) Male / female SD rats aged 2-3 months or golden hamsters aged 2-3 months were anesthetized overnight after fasting with inhaled isoflurane and given 50 IU of heparin. A PE-10 catheter was used for bile duct cannulation, and a 22-G Introcan catheter was used for portal vein cannulation. The liver was then flushed with physiological saline and UW (University of Wisconsin, UW) solution, and the liver samples collected at this time were used as control liver samples. A perfusion system was established using a circulation method, and 250 mL of Krebs-Henseleit (KH) bicarbonate buffer was prepared. Oxygenation was carried out in a mixture of 95% O2 and 5% CO2 through a fiber oxygenator to achieve an oxygen partial pressure exceeding 500 mmHg. After the liver was cold-stored at 4℃ for 48 h, samples from the cold-stored group were collected. After equilibration at room temperature for 10 min, the liver was perfused at 37℃ for 2 h, and samples perfused at room temperature after cold storage were collected. The flow rate was set to pressure control mode, and the portal vein pressure (PVP) was kept constant at 12 mmHg. The flow rate and portal vein pressure were automatically monitored and recorded. The formula for calculating portal venous resistance (PVR) is: PVR (mmHg / mL × min × g liver) = PVP (12 mmHg) / portal venous flow (mL × min × g liver). After sample collection, samples were sent to the company for metabolomics analysis. By analyzing the metabolomics data, a violin plot was created using a bioinformatics mapping website. The results are as follows: Figure 3 As shown.
[0035] Violin plots show that quinoline-4-carboxylic acid levels increased during cold storage of hamster livers and decreased during room temperature perfusion.
[0036] (4) High-passage mesenchymal stem cells (Old MSCs) with passage numbers between P10 and P15 were seeded into 96 plates. 50 μM, 100 μM, and 200 μM of QCA were added to the culture medium, respectively. Cells without added QCA served as a control. CCK8 assays were performed, and the results are as follows: Figure 4 As shown in Figure A. Low-passage MSCs (Young MSCs) with passage numbers between P4 and P7, and high-passage MSCs (Old MSCs) with passage numbers between P10 and P15, as well as high-passage MSCs treated with 50 μM and 200 μM QCA for 72 h respectively, were subjected to Ki67 immunofluorescence staining. The results are as follows. Figure 4 As shown in B.
[0037] CCK8 assay and Ki67 immunofluorescence staining showed that the addition of quinoline-4-carboxylic acid to the culture medium of advanced MSCs promoted cell proliferation.
[0038] (5) TUNEL staining was performed on low-passage MSCs (Young MSCs) with passage numbers between P4 and P7, high-passage MSCs (Old MSCs) with passage numbers between P10 and P15, and on Old MSCs treated with 50 μM and 200 μM QCA for 72 h, respectively. The results are as follows: Figure 5 As shown in Figure A. Old MSCs with 50 μM QCA were continuously added to the culture medium (replaced with fresh QCA-containing medium every 2-3 days). Cells without added QCA served as a control. Cells were passaged for three consecutive generations (passaged when cells reached 80-90% confluence) before being used for β-Gal staining experiments. The results are shown in Figure A. Figure 5 As shown in B.
[0039] TUNEL and β-Gal staining showed that adding quinoline-4-carboxylic acid to Old MSC culture medium reduced nuclear DNA breaks and delayed cell senescence.
[0040] (6) After adding preservatives to the conventional fruit fly rearing medium and dispensing it, add 115 μL of QCA (100 mM, which is equivalent to 2 mg QCA / tube) to the medium. Add the same volume of 95% ethanol to the control tube. On days 20-30 and 40-50 of fruit fly rearing, switch to feed tubes containing QCA. Add 3 male W1118 fruit flies and 3 virgin W1118 fruit flies to each rearing tube for 3-5 days, for a total of 20 tubes. After about 5-6 days, remove the parent fruit flies. Adults will begin to emerge after about 10 days. At a fixed time each day, select a batch of 400 male fruit flies that have emerged within one day, put 20 of them in each tube, and label them with their names, dates and numbers, for a total of 20 tubes. Fruit flies were reared at 25℃, 50% humidity, and under 12-hour light / 12-hour darkness conditions. The number of dead fruit flies in the rearing tubes was counted and recorded daily, and the tubes were replaced every 3-4 days. Each group of fruit flies underwent a three-times independent experiment, and the recorded data were entered into statistical software for analysis. The resulting survival curves are shown in Figure A.
[0041] Then, the tube-climbing experiment was conducted, following these steps:
[0042] 1) Collect 20 male fruit flies by anesthetizing them with CO2 and place them in a horizontally placed 250mL transparent glass graduated cylinder. Mark a position approximately 17.5cm from the bottom.
[0043] 2) The experiment was conducted under ambient light, with a temperature of 22°C and a humidity of 50%. To avoid disrupting the circadian rhythm, the experiment was always conducted at the same time each day.
[0044] 3) Use sealing film to seal the top of the cylinder to prevent fruit flies from escaping.
[0045] 4) Place the camera on a tripod. Point the camera at the 190 ml mark (17.5 cm) on the 250 ml graduated test tube.
[0046] 5) Gently tap the tube 5-10 times repeatedly to move the fruit flies to the bottom surface of the test tube. At the same time, press the "record" button on the camera to record the time it takes for the fruit flies to climb to the marked height (17.5cm) within 2 minutes.
[0047] 6) Each group of fruit flies should be tested at least 5 times, with at least 2 minutes between each test.
[0048] 7) Analysis: Analyze the video of each experiment. Record the total number of fruit flies that pass through the marked line every 10 seconds, and determine the proportion of fruit flies that are above the marked line at each time point.
[0049] 8) Plot the percentage of fruit flies at each time point, analyze the performance at 120 seconds, and perform a t-test analysis on the two groups.
[0050] The results of the tube climbing experiment are as follows Figure 6 As shown in B.
[0051] Survival curves showed that quinoline-4-carboxylic acid extended the lifespan of fruit flies, and tube crawling experiments showed that quinoline-4-carboxylic acid enhanced their locomotion ability.
[0052] (7) 21-month-old C57BL6 / J mice were randomly divided into a control group and a QCA group. The control group was given 0.9% saline (males n=7, females n=12); the QCA group was given 5 mg / kg QCA (males n=9, females n=13) by gavage for 4 consecutive months. After 4 months, the fur of the mice in the Ctrl group and QCA group was photographed to observe the coat condition, dermatitis and other phenotypes. The results are as follows: Figure 7 As shown in Figure A. The experimental mouse was placed on a grip strength meter, and its tail was gently pulled, causing the mouse to grip the probe with its forelimbs and hindlimbs. The readings on the grip strength meter when the mouse exerted maximum force were recorded. The measurements were repeated three times, and the average value was taken as the mouse's arm strength. The results are shown in Figure A. Figure 7 As shown in B. Four months after gavage administration, blood was collected from the tail vein of mice for routine blood tests to determine the proportion of neutrophils (NEUs). The results are as follows. Figure 7 As shown in Figure C. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum TNF-α levels in aged mice in the QCA treatment group and the control group. The results are shown in Figure C. Figure 7 As shown in D. Mice were periodically checked for spontaneous tumors. The number of mice with spontaneous tumors in each group was recorded. The tumor incidence rate for each group was calculated by dividing the number of mice with spontaneous tumors in each group by the total number of mice in that group. The results are shown in Figure D. Figure 7 As shown in Figure E. The concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in plasma were detected using a HITACHI 7600 Series (Japan) biochemical analyzer. The results are shown in Figure E. Figure 7 F. Survival time of mice was recorded to plot survival curves, including the Ctrl group (n = 19) and the QCA group (n = 26). The results are as follows: Figure 7 As shown in G.
[0053] The results showed that quinoline-4-carboxylic acid could alleviate symptoms such as poor coat and dermatitis in aged mice; enhance arm strength (reflecting muscle strength in mice); downregulate enzyme indicators of chronic inflammation and liver damage in aged mice; and prolong the lifespan of mice.
[0054] (8) Establishment of a 2 / 3 partial hepatectomy model in aged mice (2 / 3PHx): C57BL6 / J mice older than 18 months were anesthetized with 2% isoflurane and underwent a midline abdominal incision. The left lateral lobe and midline lobe of the liver were removed using 4-0 silk, and the relevant blood vessels and bile ducts were ligated. Mice were intraperitoneally injected with 5 mg / kg QCA or an equal volume of DMSO 2 h before, 24 h, 48 h, and 72 h after the 2 / 3 PHx procedure. Mice were sacrificed at 6 h, 48 h, and 168 h after the procedure, and liver specimens and plasma were collected. The survival time of mice after surgery was recorded for plotting survival curves. The weight of mice was measured beforehand and the weight of the liver was measured after liver removal to calculate the liver-to-body weight ratio. The concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in plasma were detected using a HITACHI 7600 Series (Japan) biochemical analyzer. Mouse liver samples collected at specific time points were fixed overnight in 4% paraformaldehyde and then embedded in paraffin. 3 μm thick paraffin sections were prepared for subsequent Ki67 immunohistochemical staining. Specifically, after antigen retrieval in EDTA (pH 8.0), the sections were incubated overnight at 4°C with primary antibody, followed by incubation at 37°C for 1 hour with secondary antibody. The sections were stained with diaminobenzidine and hematoxylin, and then observed and imaged using an optical microscope. Results are as follows: Figure 8 As shown.
[0055] The results showed that QCA supplementation did not significantly increase the weight-to-body weight ratio of the regenerated liver in mice at 6 h and 168 h post-hepatectomy, but QCA supplementation significantly increased the weight-to-body weight ratio of the regenerated liver at 48 h post-hepatectomy. Figure 8 A). Furthermore, QCA reduced liver damage enzyme markers ( Figure 8 B) improved the survival rate of mice after liver resection. Figure 8 C). Immunohistochemical staining of liver tissue showed that the QCA group had more Ki67-positive cells in its liver tissue. Figure 8 D) indicates that QCA can promote liver regeneration.
[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. Application of quinoline-4-carboxylic acid in the preparation of anti-aging drugs.
2. Use according to claim 1, characterized in that, The anti-aging drugs mentioned are specifically drugs that delay the aging of mesenchymal stem cells.
3. Use according to claim 2, characterized in that, The drugs that delay the aging of mesenchymal stem cells are specifically drugs that promote the proliferation of mesenchymal stem cells.
4. Use according to claim 2, characterized in that, The drugs that delay the aging of mesenchymal stem cells are specifically drugs that reduce damage to the nuclear DNA of mesenchymal stem cells.
5. The use according to claim 1, characterized in that, The anti-aging drugs mentioned are specifically drugs that enhance athletic ability and muscle strength.
6. Application of quinoline-4-carboxylic acid in the preparation of drugs that promote liver regeneration.
7. Application of quinoline-4-carboxylic acid in the preparation of drugs to improve post-hepatectomy survival rates.
8. The use according to any one of claims 6 to 7, characterized in that, The drugs that promote liver regeneration and the drugs that improve post-hepatectomy survival rates are specifically drugs that reduce liver inflammation.
9. The use according to any one of claims 6 to 7, characterized in that, The drugs that promote liver regeneration and the drugs that improve post-hepatectomy survival rates are specifically drugs that reduce liver damage.