Application of quinoline-4-carboxylic acid in preparation of Anti-aging products, and liver protection and liver regeneration promotion products

Abstract

The present invention provides application of quinoline-4-carboxylic acid in the preparation of anti-aging products, and liver protection and liver regeneration promotion products, and belongs to the technical field of medicines. The present invention reveals that quinoline-4-carboxylic acid can be used as a novel additive for in vitro expansion culture of mesenchymal stem cells, which breaks through the bottleneck that mesenchymal stem cells are prone to aging in vitro culture, and optimizes the clinical development and application of mesenchymal stem cells; quinoline-4-carboxylic acid can relieve the aging-related phenotype of drosophila and mouse, enhance the movement ability of the drosophila and the muscle strength of the mouse, prolong the life span of the drosophila and the mouse, and thus is expected to become an effective component of anti-aging treatment drugs or health-care products; quinoline-4-carboxylic acid can increase the survival rate of mice after the liver cutting operation, promote liver regeneration after liver cutting injury, and thus is expected to become an effective component of drugs or health-care products to promote the prognosis of patients after the liver cutting operation, to promote liver regeneration, and to relieve or treat liver injury.

Description

Application of quinoline-4-carboxylic acid in the preparation of anti-aging products and products for protecting the liver and promoting liver regeneration Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the application of quinoline-4-carboxylic acid in the preparation of anti-aging products and products for protecting the liver and promoting liver regeneration. Background Art

[0002] Aging is characterized by a progressive loss of physiological integrity, leading to functional impairment and an increased risk of death. For decades, biologists have been faced with the challenge of how to delay aging and extend lifespan. Most hibernating animals live longer than their non-hibernating counterparts, and they exhibit excellent metabolic adaptation and damage protection in extreme environments. Therefore, hibernating animal models are excellent models for studying metabolism, hypoxia / reperfusion, and longevity. As a hibernating animal, the thirteen-striped hamster can tolerate low temperatures and adapt to the cold by reducing the cold sensitivity of the peripheral somatosensory system and preventing cell damage caused by reactive oxygen species. Similarly, the thirteen-striped hamster's induced pluripotent stem cells (GS iPSC) also show a stronger ability to adapt to stressful environments. Using the GS iPSC cell model, the cold adaptation protection mechanism of hibernating animals is studied from a metabolic perspective, in order to apply it to the anti-aging field to solve human aging-related diseases.

[0003] Quinoline-4-carboxylic acid (QCA) is a quinoline monocarboxylic acid discovered through freeze-thaw metabolomics analysis of GS iPSC cells. Existing data suggest 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, antimicrobial, and antifungal properties. Furthermore, numerous studies suggest that quinoline derivatives have protective effects against oxidative stress, alleviating age-related oxidative damage in rats. However, due to limited available literature, whether QCA exerts protective effects in aging and regeneration models remains unclear.

[0004] Mesenchymal stem cells (MSCs) are a type of multipotent stem cell derived from the mesoderm with multipotent differentiation potential. They are an important member of the stem cell family. Currently, bone marrow- and umbilical cord-derived MSCs are the most widely studied. Due to their multipotential differentiation and diverse cytokine secretion, MSCs have found widespread clinical application, primarily in the treatment of neurological damage, liver disease, myocardial ischemia, diabetes, and skin conditions. MSCs are the most widely used of the marketed stem cell products. With increasing research, scientists have discovered that, in addition to their multipotential differentiation, MSCs can also promote stem cell engraftment, hematopoietic support, immune regulation, and self-replication, making them a hot topic in this field. The number of cells isolated from donors is typically limited. To obtain sufficient cells for clinical treatment, MSCs must be expanded and cultured in vitro. The tendency of MSCs to age after long-term in vitro culture has hindered their clinical development and application. Therefore, it is crucial to understand the mechanisms underlying MSC aging, how to mitigate MSC aging, and to develop safe and effective anti-aging supplements for in vitro culture.

[0005] As the center of human metabolism, the liver plays a vital role in numerous physiological and pathological processes, and numerous diseases can affect the liver. Partial liver resection is currently the most common and effective treatment for liver tumors. The robust regenerative capacity of normal liver tissue underlies effective liver resection. However, due to limitations in early liver tumor diagnosis, most liver tumors are already at advanced stages by the time of initial diagnosis. Due to the large size of the tumors and the involvement of multiple liver segments, the volume of the remaining liver after resection is insufficient to meet the body's metabolic needs, leading to life-threatening complications such as liver failure. To address this issue, various surgical approaches have been developed to stimulate rapid regeneration of the remaining liver, including portal vein ligation (PVL) / portal vein embolization (PVE) and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS). However, the effectiveness of these procedures remains controversial, and the risk of liver failure after surgery due to insufficient liver remnant volume 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 a variety of diseases, and how to delay aging has always been a hot topic for scientists. Stem cells play an important role in the aging process, and the aging of stem cells accelerates the aging of the human body. Mesenchymal stem cells (MSCs) are stem cells with self-replication and multidirectional differentiation potential. Although the existing MSCs in vitro expansion culture medium can allow them to expand and culture for several generations in vitro, it still fails to achieve satisfactory results in delaying cell aging. As mentioned above, the effectiveness of existing surgeries to stimulate rapid regeneration of residual liver is still controversial, and the risk of liver failure in patients after surgery due to insufficient residual liver volume is still high. Therefore, further research and development of new technical solutions or drugs to delay aging (stem cell aging) and promote liver regeneration after liver resection are needed to lay the foundation for clinical application.

[0007] Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides the use of quinoline-4-carboxylic acid in the preparation of anti-aging products and products for protecting the liver and promoting liver regeneration. Specifically, quinoline-4-carboxylic acid can be used as a new additive for the in vitro expansion and culture of mesenchymal stem cells, breaking through 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 the aging-related phenotypes of fruit flies and mice, enhance the motor 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 for anti-aging drugs or health products; quinoline-4-carboxylic acid can improve the survival rate of mice after liver resection and promote liver regeneration after liver resection injury, and is expected to become an effective ingredient for drugs or health products that improve the prognosis of patients after liver resection, promote liver regeneration, and alleviate or treat liver damage.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] One of the purposes of the present invention is to provide the use of quinoline-4-carboxylic acid in the preparation of anti-aging products.

[0011] Furthermore, the anti-aging product is specifically a product that delays the aging of mesenchymal stem cells.

[0012] Furthermore, the product for delaying the aging of mesenchymal stem cells is specifically a product for promoting the proliferation of mesenchymal stem cells and / or a product for reducing DNA damage in the nucleus of mesenchymal stem cells.

[0013] Furthermore, the anti-aging product is specifically a product that enhances athletic ability and muscle strength.

[0014] The second purpose of the present invention is to provide the use of quinoline-4-carboxylic acid in the preparation of liver protection products.

[0015] The third object of the present invention is to provide the use of quinoline-4-carboxylic acid in the preparation of a product for promoting liver regeneration.

[0016] A fourth object of the present invention is to provide the use of quinoline-4-carboxylic acid in the preparation of a product for improving the survival rate after liver resection.

[0017] Furthermore, the liver protection product, the liver regeneration promotion product and the post-hepatectomy survival rate improvement product are specifically products for alleviating liver inflammation and / or products for alleviating liver damage.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] Using hibernating animals as a natural model, the inventors innovatively utilized induced pluripotent stem cells (iPSCs) from the hibernating ground squirrel (Sciurus thirteen-striped ground squirrel) to explore the molecular mechanisms and metabolic regulation during cold acclimation and rewarming in hibernating animals. They discovered that quinoline-4-carboxylic acid (Q4C) increases during cold acclimation and decreases during rewarming, identifying it as a metabolite that can mitigate stress-induced damage. This potential application suggests that Q4C can be used to combat age-related damage, delay aging, and promote regeneration and repair. Specific experiments have shown that adding Q4C to the culture medium of mesenchymal stem cells in vitro promotes the proliferation of aged mesenchymal stem cells, reduces nuclear DNA fragmentation, and slows aging. Q4C can also alleviate symptoms such as poor coat quality and dermatitis in aged mice. Q4C can also downregulate enzyme markers of chronic inflammation and liver damage. Q4C can enhance arm strength and prolong lifespan in aged mice. Q4C can also enhance the motor ability and lifespan of fruit flies. Q4C can also promote liver regeneration and improve survival after hepatectomy in aged mice, alleviating enzymatic markers of liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 shows the nuclear DNA fragmentation of frozen-thawed GS iPSCs and hMSCs in Example 1 of the present invention;

[0021] FIG2 is a volcano plot and violin plot of quinoline-4-carboxylic acid in the freeze-thaw metabolome of GS iPSC cells in Example 1 of the present invention;

[0022] FIG3 is a violin plot showing changes in the content of quinoline-4-carboxylic acid in the hamster liver cold storage-normothermic perfusion metabolome according to Example 1 of the present invention;

[0023] FIG4 shows the effect of adding different concentrations of quinoline-4-carboxylic acid to a high-generation mesenchymal stem cell culture medium on cell proliferation in Example 1 of the present invention;

[0024] FIG5 shows the nuclear DNA fragmentation and reduction of senescent cells in high-generation mesenchymal stem cells after treatment with quinoline-4-carboxylic acid in Example 1 of the present invention;

[0025] FIG6 shows the survival curve and tube climbing test results of Drosophila after adding quinoline-4-carboxylic acid in Example 1 of the present invention;

[0026] FIG7 shows changes in coat, dermatitis, arm strength, chronic inflammation, and enzymatic indicators of liver damage in elderly mice after addition of quinoline-4-carboxylic acid in Example 1 of the present invention;

[0027] FIG8 shows the effect of adding quinoline-4-carboxylic acid in Example 1 of the present invention on the ratio of regenerated liver weight to body weight, survival rate after hepatectomy, and enzymatic indicators of liver damage in mice. DETAILED DESCRIPTION

[0028] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are intended to be within the scope of the present invention. The equipment and raw materials used in the following examples are all commercially available, and the methods used in the examples are consistent with conventional methods unless otherwise specified.

[0029] The technical solution of the present invention is further elaborated in detail below in conjunction with embodiments.

[0030] Example 1

[0031] (1) GS iPSCs or hMSCs cultured under normal conditions were used as 37°C control samples. For cold exposure treatment, the culture medium was replaced with UW solution. After being placed at room temperature for 10 minutes, the cells replaced with UW solution were transferred to a 4°C refrigerator and stored for 48 hours. For rewarming treatment, the cells were taken out of the refrigerator and replaced with pre-cooled culture medium. After being placed at room temperature for 10 minutes, the cells were transferred to a 37°C incubator for 2 hours and then collected at 4°C for 4 hours and 37°C for 2 hours. The cells were fixed with 4% paraformaldehyde and then subjected to TUNEL staining. The staining results are shown in Figure 1.

[0032] The results showed that after the cells were frozen and thawed, TUNEL staining showed that the number of nuclear DNA breaks in hMSCs increased compared with GS iPSCs, suggesting that GS iPSCs have stronger adaptability to stress environments.

[0033] (2) GS iPSCs cultured under normal conditions were used as samples for the 37°C control group. For cold exposure treatment, the GS iPSC culture medium was replaced with Hibernate-A culture medium. After being placed at room temperature for 10 minutes, the cells replaced with Hibernate-A culture medium were transferred to a 4°C refrigerator and stored for 4 hours, and then the 4°C 4 hour samples were collected. For rewarming treatment, the cells were taken out of the refrigerator and replaced with pre-cooled GS iPSC culture medium. After being placed at room temperature for 10 minutes, the cells were transferred to a 37°C incubator for 2 hours, and then the 4°C 4 hour and 37°C 2 hour 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 made using the bioinformatics mapping website. The results are shown in Figure 2.

[0034] Both the volcano plot and the violin plot showed that quinoline-4-carboxylic acid increased during cold acclimation and decreased during rewarming.

[0035] (3) Male / female SD rats or golden hamsters aged 2-3 months were fasted overnight and anesthetized with isoflurane inhalation and given 50 IU heparin. The bile duct was cannulated with a PE-10 catheter, and the portal vein was cannulated with a 22-G introcan catheter. The liver was then flushed with normal saline and UW (University of Wisconsin, UW) solution, and the liver was collected at this time as the control liver sample. A perfusion system was established using a circulation method, and 250 mL of Krebs-Henseleit (KH) bicarbonate buffer was prepared. Oxygenation was performed using a fiber oxygenator in a mixture of 95% O2 and 5% CO2 to a partial pressure of oxygen exceeding 500 mmHg. The liver was cold-stored at 4°C for 48 hours, and then the cold-stored group sample was collected. The liver was equilibrated at room temperature for 10 minutes, and then perfused at 37°C for 2 hours before the cold-stored and normothermic perfused sample was collected. The flow rate was set to pressure control mode, and the portal vein pressure (PVP) was constant at 12 mmHg. The flow rate and portal vein pressure were automatically monitored and recorded. The formula for calculating portal vein resistance (PVR) is: PVR (mmHg / mL × min × g liver) = PVP (12 mmHg) / portal vein flow (mL × min × g liver). After sample collection, the samples were sent to the company for metabolomics analysis. Violin plots were generated using the Bioinformatics Plotting website, as shown in Figure 3.

[0036] Violin plots showed that quinoline-4-carboxylic acid increased during cold storage and decreased during normothermic perfusion in hamster liver.

[0037] (4) High-passage mesenchymal stem cells (Old MSCs) with passage numbers between P10 and 15 were plated in 96-well plates. 50 μM, 100 μM, and 200 μM QCA were added to the culture medium, respectively. A control without QCA was used for CCK8 assays. The results are shown in Figure 4A. Ki67 immunofluorescence staining was performed on low-passage MSCs (Young MSCs) with passage numbers between P4 and 7, high-passage MSCs (Old MSCs) with passage numbers between P10 and 15, and high-passage MSCs treated with 50 μM and 200 μM QCA for 72 h, respectively. The results are shown in Figure 4B.

[0038] CCK8 assay and Ki67 immunofluorescence staining showed that the addition of quinoline-4-carboxylic acid to high-generation MSC culture medium could promote cell proliferation.

[0039] (5) TUNEL staining was performed on young MSCs (P4-7), old MSCs (P10-15), and old MSCs treated with 50 μM and 200 μM QCA for 72 h, respectively. The results are shown in Figure 5A. Old MSCs were continuously supplemented with 50 μM QCA (fresh QCA-containing medium was replaced every 2-3 days). Cells without QCA served as a control. After three consecutive passages (passaged when the cells reached 80-90% confluence), they were used for β-Gal staining. The results are shown in Figure 5B.

[0040] TUNEL staining and β-Gal staining showed that the addition of quinoline-4-carboxylic acid to the culture medium of Old MSCs could reduce nuclear DNA fragmentation and delay cell senescence.

[0041] (6) After adding preservatives to the conventional fruit fly breeding medium and packaging, 115 μL of QCA (100 mM, equivalent to 2 mg QCA / tube) was added to the medium, and the same volume of 95% ethanol was added to the control tube. On the 20th to 30th day and the 40th to 50th day of fruit fly breeding, the feed tube containing QCA was replaced for breeding. Three male W1118 fruit flies and three virgin W1118 fruit flies were added to each feeding tube and bred for 3-5 days, for a total of 20 tubes. After about 5-6 days, the parent fruit flies were removed, and adults began to emerge about 10 days later. At a fixed time every day, a round of male fruit flies within 1 day of eclosion were selected, totaling 400, and 20 were placed in each tube. The names, dates, and numbers of flies were marked, for a total of 20 tubes. Fruit flies were reared at 25°C, 50% humidity, and a 12-hour light / 12-hour dark cycle. The number of flies that died within the tubes was counted and recorded daily, and the tubes were replaced every 3-4 days. Each group of fruit fly lifespan tests was repeated three times, and the data were analyzed using statistical software. The resulting survival curve is shown in Figure A.

[0042] Then carry out the tube climbing experiment, the steps are as follows:

[0043] 1) Collect 20 male fruit flies anesthetized with CO2 and place them in a horizontal 250 mL clear glass measuring cylinder. Mark the cylinder approximately 17.5 cm from the bottom.

[0044] 2) Experiments were performed under ambient light, with a temperature of 22°C and a humidity of 50%, respectively. To avoid confusion with circadian rhythms, experiments were always performed at the same time each day.

[0045] 3) Seal the top of the cylinder with parafilm to prevent fruit flies from escaping.

[0046] 4) Place the camera on a tripod and aim the camera at the 190 ml line (17.5 cm) on the 250 ml graduated test tube.

[0047] 5) Repeatedly tap gently 5-10 times to move the fruit fly to the bottom surface of the test tube. At the same time, press the "record" button on the camera to record the fruit fly climbing to the marked height (17.5 cm) within 2 minutes.

[0048] 6) Repeat the experiment at least 5 times for each group of fruit flies, with at least 2 minutes between each experiment

[0049] 7) Analysis: Analyze the video of each experiment. Every 10 seconds, record the total number of fruit flies passing through the marked line and calculate the proportion of fruit flies above the marked line at each time point.

[0050] 8) Plot the percentage of flies at each time point, analyze the performance at 120 seconds, and perform a t-test analysis on the two groups.

[0051] The results of the tube climbing experiment are shown in Figure 6B.

[0052] The survival curve showed that quinoline-4-carboxylic acid extended the lifespan of Drosophila, and the tube crawling experiment showed that quinoline-4-carboxylic acid enhanced its locomotion ability.

[0053] (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 (male mice n=7, female mice n=12); the QCA group was given 5 mg / kg QCA (male mice n=9, female mice n=13) for four consecutive months. Four months later, the hair of the mice in the Ctrl and QCA groups was photographed to observe the coat condition, dermatitis and other phenotypes of the mice. The results are shown in Figure 7A. The experimental mice were placed on a grip strength meter. The tail of the mouse was gently pulled so that the mouse's forelimbs and hindlimbs grasped the probe with force. The reading on the grip strength meter when the mouse exerted the maximum force was recorded. The measurement was repeated three times and the average value of the mouse's arm strength was taken. The results are shown in Figure 7B. Four months after gavage administration, the tail vein blood of the mice was collected and the proportion of neutrophils (NEU) was determined by routine blood testing. The results are shown in Figure 7C. The enzyme-linked immunosorbent assay (ELISA) was used to detect the content of TNF-α in the serum of the elderly mice in the QCA treatment group and the control group. The results are shown in Figure 7D. Mice were regularly examined for the development of spontaneous tumors. The number of mice with spontaneous tumors in each group was recorded. The tumor incidence rate in each group was calculated by dividing the number of mice with spontaneous tumors by the total number of mice in each group. The results are shown in Figure 7E . Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) concentrations were measured using a HITACHI 7600 Series (Japan) biochemical analyzer. The results are shown in Figure 7F . Survival time of mice was recorded and survival curves were constructed. The results for the Ctrl group (n = 19) and the QCA group (n = 26) are shown in Figure 7G .

[0054] The results showed that quinoline-4-carboxylic acid can alleviate symptoms such as poor coat and dermatitis in elderly mice; enhance the arm strength of elderly mice (reflecting the muscle strength of mice), downregulate enzymatic indicators of chronic inflammation and liver damage; and prolong the lifespan of mice.

[0055] (8) Establishment of 2 / 3 partial hepatectomy model in elderly mice (2 / 3PHx): C57BL6 / J mice older than 18 months were anesthetized by inhalation of 2% isoflurane and then underwent a midline abdominal incision. The left lateral lobe and median 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 2 / 3PHx surgery and 24 h, 48 h, and 72 h after surgery. Mice were killed at 6 h, 48 h, and 168 h after surgery, and liver specimens and plasma were collected. The survival time of mice after surgery was recorded for the purpose of drawing survival curves. The mice were weighed in advance and the liver weight was weighed after liver removal to calculate the liver weight to body weight ratio. The concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in plasma were measured 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. Following antigen retrieval in EDTA (pH 8.0), sections were incubated with the primary antibody overnight at 4°C, followed by incubation with the secondary antibody at 37°C for 1 hour. Sections were stained with diaminobenzidine and hematoxylin, and then observed and imaged using a light microscope. The results are shown in Figure 8.

[0056] The results showed that QCA supplementation did not significantly increase the regenerated liver weight to body weight ratio in mice when samples were collected 6 and 168 hours after hepatectomy. However, QCA supplementation significantly increased the regenerated liver weight to body weight ratio 48 hours after hepatectomy (Figure A). Furthermore, QCA reduced enzymatic markers of liver damage (Figure B) and improved post-hepatectomy survival in mice (Figure C). Immunohistochemical staining of liver tissue for Ki67 revealed an increased number of Ki67-positive cells in the QCA group (Figure D), indicating that QCA promotes liver regeneration.

[0057] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of quinoline-4-carboxylic acid in the preparation of anti-aging products.

2. The use according to claim 1, It is characterized in that The anti-aging product is specifically a product that delays the aging of mesenchymal stem cells.

3. The use according to claim 2, It is characterized in that The product for delaying the aging of mesenchymal stem cells is specifically a product for promoting the proliferation of mesenchymal stem cells.

4. The use according to claim 2, It is characterized in that The product for delaying the aging of mesenchymal stem cells is specifically a product for reducing DNA damage in the nucleus of mesenchymal stem cells.

5. The use according to claim 1, It is characterized in that The anti-aging product is specifically a product that enhances athletic ability and muscle strength.

6. Application of quinoline-4-carboxylic acid in the preparation of liver protection products.

7. Application of quinoline-4-carboxylic acid in the preparation of products for promoting liver regeneration.

8. Application of quinoline-4-carboxylic acid in the preparation of products for improving survival rate after liver resection.

9. The use according to any one of claims 6 to 8, It is characterized in that The liver protection product, the liver regeneration promotion product and the survival rate improvement product after liver resection are specifically products that reduce liver inflammation.

10. The use according to any one of claims 6 to 8, It is characterized in that The liver protection product, the liver regeneration promotion product and the survival rate improvement product after liver resection are specifically products that reduce liver damage.

Images