Compounds targeting tdo2 and their use for delaying liver aging

Abstract

The present disclosure relates to compounds targeting TDO2 and their use for delaying liver aging. Specifically, the present disclosure found that TDO2 levels are lower in elderly subjects than in younger subjects. TDO2 levels are significantly downregulated with age. TDO2 knockout mice exhibit more severe liver aging phenotypes; mice have decreased ability to degrade toxins and accumulate more toxins in the liver; levels of AHR, PXR, CAR, CYPs are significantly downregulated. Supplementation with lactic acid increases TDO2 levels, reduces liver aging, and decreases ROS levels.

Description

Technical Field

[0001] This application relates to the biological, medical, and clinical fields. Specifically, it relates to the use of lactic acid in increasing the body's TDO2 levels and in the prevention and treatment of liver aging. Background Technology

[0002] The global population is aging rapidly. By 2030, older people (generally defined as those aged 65 and over) are expected to account for more than 25% of the world's total population, and the burden of age-related diseases is increasing.

[0003] As a core organ for metabolism and detoxification, the liver's aging process has a profound impact on overall health. Liver aging not only involves the natural decline of hepatocyte function but also reduces the body's ability to clear exogenous and endogenous harmful substances, increasing susceptibility to liver diseases. It has been reported that hepatocyte aging promotes lipid accumulation and induces hepatic steatosis. Clearing senescent cells (such as p16Ink4a-positive cells) or using drugs that target and clear senescent cells can significantly alleviate hepatic steatosis. Furthermore, liver aging also involves the accumulation of senescent hepatocytes, sinusoidal endothelial cells, hepatic stellate cells, and Kupffer cells, creating a pro-inflammatory and pro-fibrotic microenvironment. This type of cellular senescence is characterized by irreversible cell cycle arrest and the secretion of aging-associated phenotypic factors (SASPs), which exacerbate chronic inflammation and tissue damage, promoting liver fibrosis and even tumor development.

[0004] In recent years, the core regulatory mechanisms of liver aging have received widespread research attention. Liver detoxification mainly relies on various enzyme systems, with the cytochrome P450 (CYP) family and antioxidant enzymes being particularly crucial. Among these, the three types of exogenous receptors—aromatic hydrocarbon receptors (AhR), pregnane X receptors (PXR), and constitutive androstenedione receptors (CAR)—have evolved to regulate hepatocyte detoxification pathways by transcribedly activating cytochrome P450 enzymes (CYPs) and other detoxification enzymes. Currently, a large number of synthetic substances enter the liver for metabolism via the portal vein, making liver detoxification highly dependent on these exogenous receptors, thus their role is becoming increasingly critical. Therefore, maintaining high expression levels of AhR, PXR, and CAR is essential for clearing excess exogenous toxins, and this mechanism holds promise for protecting liver physiological function and delaying liver aging.

[0005] Tryptophan 2,3-dioxygenase (TDO2) is a key rate-limiting enzyme catalyzing the conversion of tryptophan to kynurenine, participating in multiple physiological processes such as immune homeostasis regulation, neural signal transduction, and tumor microenvironment remodeling. This protein is mainly expressed in the liver. However, a puzzling phenomenon is that its isoenzyme, indoleamine 2,3-dioxygenase (IDO1), is widely expressed in most tissues and organs throughout the body. This difference has sparked academic discussion: why did the liver selectively utilize TDO2, rather than IDO1, to catalyze the same chemical reaction during evolution? Notably, the half-life of TDO2 protein is only about 2 hours, and its degradation rate is much faster than the average half-life of total liver protein. This characteristic suggests that TDO2 may possess independent biological functions beyond enzymatic catalysis. Summary of the Invention

[0006] The inventors unexpectedly discovered that TDO2 can act as a transcription factor in hepatocytes, transactivating exogenous receptors such as AhR, PXR, and CAR, thereby regulating the liver detoxification pathway; targeted upregulation of TDO2 expression can delay liver aging. This disclosure also reveals that lactate can directly act on hepatocytes, upregulating TDO2 expression; exogenous lactate intervention experiments showed that the detoxification capacity of hepatocytes in mouse models and human liver organoids was significantly enhanced, effectively delaying the liver aging process.

[0007] New pharmaceutical uses of lactic acid According to some embodiments of this disclosure, the use of lactic acid in the preparation of a medicament for improving hepatic aging in mammals is provided, wherein the medicament is selected from any one or a combination of the following: Upregulates hepatic tryptophan 2,3-dioxygenase (TDO2) expression, Reduce liver ROS levels Reduce the proportion of SA-β-Gal positive cells in the liver Reduce the proportion of p16Ink4a-positive cells in the liver.

[0008] In some implementation schemes, the mammals are selected from: humans, mice, rats, guinea pigs, rabbits, horses, monkeys, and dogs.

[0009] In some implementations, the use of lactic acid in the preparation of drugs that improve human liver aging is provided.

[0010] In some embodiments, the drug is prepared as an oral or injectable formulation. The oral formulation is selected from: solutions, suspensions, tablets, capsules, granules, powders, enteric-coated formulations, sustained-release formulations, and controlled-release formulations. The injectable formulation is selected from: injection solutions and lyophilized powders for injection.

[0011] In some embodiments, the medicament of this disclosure can be conveniently presented in a unit dosage form, associating the active ingredient with a drug carrier or excipient. In some embodiments, the medicament of this disclosure further includes one or more pharmaceutically acceptable carriers, excipients, or other inactive ingredients.

[0012] New Diagnostic Uses of TDO2 According to some embodiments of this disclosure, the use of a TDO2 detection reagent in the preparation of a kit is provided, the kit being used to characterize the degree of liver aging in a subject; the TDO2 detection reagent is capable of quantifying the TDO2 expression level in a subject's liver tissue sample.

[0013] In some implementations, the expression level is the nucleic acid expression level or the protein expression level.

[0014] In some implementations, when the expression level is a nucleic acid expression level, the TDO2 detection reagent comprises: a TDO2-specific primer or a TDO2-specific probe.

[0015] TDO2-specific primers are artificially designed and synthesized DNA fragments targeting the specific nucleic acid sequence of the TDO2 gene. They are targeted reagents for nucleic acid amplification technologies such as polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR). TDO2-specific primers bind specifically to the TDO2 gene template region in the sample through complementary base pairing, initiating the specific amplification of the TDO2 gene fragment.

[0016] TDO2-specific probes are single-stranded nucleic acid probes (primarily DNA probes) designed to target the specific conserved sequence of the TDO2 gene and labeled with fluorescent or biotinylate groups. They are mainly compatible with detection technologies such as real-time quantitative PCR, in situ hybridization, and Western blotting. The probes specifically recognize and bind to the TDO2 target nucleic acid fragment during amplification, releasing detectable fluorescent or colorimetric signals through the labeled groups. This enables real-time monitoring of the TDO2 gene amplification process and quantification of the TDO2 gene copy number and expression level in the sample.

[0017] Those skilled in the art can easily prepare TDO2-specific primers or probes. The TDO2 gene sequence information is publicly available and highly conserved. The complete TDO2 gene sequence and conserved coding region sequences have been fully entered into public gene databases such as NCBI, GenBank, and Ensembl, allowing technicians to directly obtain TDO2 targeting sequence templates. Primer and probe design techniques are mature and standardized. Those skilled in the art can use software such as Primer Premier, OligoCalc, Vector NTI, and Primer-BLAST, based on the conserved TDO2 gene sequence, to complete primer specificity screening, annealing temperature adaptation, primer dimer selection, hairpin structure analysis, and non-specific binding site screening. Simultaneously, they can optimize probe length, design labeling sites, and verify specificity.

[0018] In some embodiments, when the expression level is a protein expression level, the TDO2 detection reagent comprises: a TDO2-specific antibody or an antigen-binding fragment thereof.

[0019] In some implementations, as examples, the antibody is selected from: mouse antibodies, chimeric antibodies, and humanized antibodies. In some implementations, examples of antigen-binding fragments include, but are not limited to: Fab, Fab′, F(ab′)2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.

[0020] Once those skilled in the art know the sequence information of the target, they can design specific antibodies against a specific epitope based on known technologies.

[0021] In some implementation schemes, when the TDO2 expression level of the first subject or the first subject group is statistically significantly higher than that of the second subject or the second subject group, the liver aging degree of the first subject or the first subject group is determined to be lower than that of the second subject or the second subject group.

[0022] In other implementations, if the TDO2 expression level of the first subject or the first subject group is statistically significantly lower than that of the second subject or the second subject group, then the liver aging degree of the first subject or the first subject group is determined to be higher than that of the second subject or the second subject group.

[0023] In other implementations, if the TDO2 expression level of the first subject or the first subject group is statistically significantly higher than the TDO2 expression level of the second subject or the second subject group, then the age of the first subject or the first subject group is determined to be lower than that of the second subject or the second subject group.

[0024] In other implementations, if the TDO2 expression level of the first subject or the first subject group is statistically significantly lower than the TDO2 expression level of the second subject or the second subject group, then the age of the first subject or the first subject group is determined to be higher than that of the second subject or the second subject group.

[0025] In this disclosure, ordinal numbers such as "first" and "second" are used only to distinguish different technical features, components, steps, and compositions, and are not intended to limit the quantity, order, level, or importance. The ordinal qualifiers such as "first" and "second" used in this disclosure are only used to distinguish and define different technical features, functional modules, structural elements, component raw materials, number of device parts, and other similar technical objects. These ordinal qualifiers do not have the limiting meaning of quantity, execution order, hierarchical level, degree of importance, priority, spatial arrangement order, etc., and should not be interpreted as constraining the quantity, operation order, superiority or inferiority level, or core or secondary attributes of the corresponding technical objects.

[0026] When the kit is used to determine the nucleic acid expression level of a sample to be tested, in addition to the TDO2 detection reagent, the kit contains any one or a combination of the following: RNA preservation reagent, RNA extraction reagent, reverse transcription reagent, sequencing buffer, PCR amplification reagent, nucleic acid purification reagent, hybridization buffer, and sequencing library construction reagent.

[0027] As an example of an application scenario, RNA extraction reagents and nucleic acid purification reagents are used to isolate and purify TDO2 target nucleic acids from test samples, removing interference from impurities. Reverse transcription reagents enable the reverse transcription conversion of RNA to cDNA, adapting to subsequent amplification and detection needs. PCR amplification reagents provide the reaction system for the specific amplification of target nucleic acid fragments.

[0028] When the kit is used to determine the protein expression level of a sample, in addition to the TDO2 detection reagent, the kit contains any one or a combination of the following: ELISA coating buffer, ELISA washing buffer, ELISA stop solution, protein loading buffer, protein ladder, electrophoresis buffer, transfer buffer, paraformaldehyde tissue fixation solution, antigen retrieval solution, permeabilization solution, blocking solution, fluorescent secondary antibody, enzyme-labeled secondary antibody, chromogenic substrate, luminescent substrate, and mass spectrometry sample processing reagent.

[0029] As an example of application scenarios, such as immunohistochemistry, paraformaldehyde tissue fixative, antigen retrieval solution, and permeabilization solution are used for the initial fixation, retrieval, and permeabilization of tissue samples to expose the TDO2 antigen sites in the sample. Blocking solution can block non-specific binding sites, reducing experimental background interference. In ELISA applications, ELISA coating solution, ELISA washing solution, ELISA stop solution, enzyme-labeled secondary antibody, and chromogenic / luminescent substrates are adapted for enzyme-linked immunosorbent assays (ELISA) to achieve quantitative protein detection. Protein loading buffer, protein ladder, electrophoresis and transfer buffer are adapted for protein electrophoresis and Western blot detection for protein separation and qualitative analysis.

[0030] Methods and applications in the preparation of animal models According to some embodiments of this disclosure, the use of TDO2 knockout reagents or TDO2 knockdown reagents in the preparation of animal models, wherein the animal models are liver aging models for drug screening.

[0031] According to some embodiments of this disclosure, a method for constructing a liver aging model is provided. Methods known in the art for preparing gene knockout animals are also applicable to the method of this disclosure. As an example, TDO2 knockout reagents or TDO2 knockdown reagents are introduced into experimental animals using conventional methods such as microinjection, tail vein injection, in situ injection, or liposome transfection delivery, targeting the animal's liver tissue. For embryonic knockout models, TDO2 knockout reagents can be introduced into embryonic cells via pronuclear microinjection of fertilized eggs, followed by embryo transfer to obtain systemic or liver-specific TDO2 gene knockout.

[0032] In some implementations, the animal is a rodent.

[0033] In some implementations, the animal is selected from mice, rats, rabbits, and guinea pigs.

[0034] In some implementations, the mice are selected from the following strains: C57BL / 6J, C57BL / 6N, BALB / c, DBA / 2, ICR, and KM.

[0035] In some implementations, the model presents with symptoms or combinations of the following: liver fibrosis, hepatic steatosis, reduced liver capacity to clear toxins, decreased AHR expression levels in liver tissue, decreased PXR expression levels in liver tissue, decreased CAR expression levels in liver tissue, decreased CYP expression levels in liver tissue, decreased NADPH expression levels in liver tissue, and glycogen accumulation.

[0036] In some implementations, the candidate drug is administered to the animal model, and if the expression level or activity of TDO2 in the liver tissue increases (nucleic acid level or protein level) compared to before the administration of the candidate drug, the candidate drug is determined to improve liver aging.

[0037] In some implementations, if the expression level or activity of TDO2 in liver tissue does not change significantly or decreases (nucleic acid level or protein level) when the candidate drug is administered to the animal model compared to before the administration of the candidate drug, then the candidate drug is determined to have limited ability to improve liver aging or liver aging.

[0038] In some embodiments, the TDO2 knockout reagent comprises any one or a combination of the following: a Cas9 nuclease or a Cas9 expression vector, a TDO2-specific sgRNA or its expression plasmid, a donor template, and a Cre-loxP element. In some specific embodiments, the TDO2 knockout reagent comprises at least: a Cas9 nuclease or a Cas9 expression vector, a TDO2-specific sgRNA or its expression plasmid.

[0039] In some embodiments, the TDO2 knockdown reagent comprises any one or a combination of the following: antisense oligonucleotides, siRNA, dsRNA, esiRNA, shRNA, CRISPRi. Attached Figure Description

[0040] Figures 1A to 1B The study showed that TDO2 expression in the liver was reduced in older adults.

[0041] Figure 2 The study showed that TDO2 levels in the livers of older mice were decreased.

[0042] Figures 3A to 3B The study showed that TDO2 deficiency leads to increased aging in mice, and that increasing TDO2 levels helps reduce liver aging.

[0043] Figure 4 The results showed that TDO2 knockout mice had a reduced ability to clear toxins from their livers and accumulated more toxins.

[0044] Figure 5 The results show that TDO2 knockout leads to a decrease in the levels of AHR, PXR, CAR and their downstream CYPs.

[0045] Figures 6A to 6B The results showed that TDO2 deficiency led to glycogen accumulation and a decrease in NADPH.

[0046] Figures 7A to 7B The results showed that lactic acid can increase TDO2 levels, reduce liver aging, and lower ROS levels. Detailed Implementation

[0047] Experimental materials: Male wild-type C57BL / 6 mice were purchased from the Laboratory Animal Center, Chinese Academy of Medical Sciences (Beijing, China). These animals were housed under sterile conditions at the animal facilities of the Chinese Academy of Medical Sciences. All studies involving mice were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences (ACUC-A02-2025-033).

[0048] Liver samples were taken from normal liver tissue regions of donors, provided by the National Developmental and Functional Human Brain Tissue Resource Bank of the Chinese Academy of Medical Sciences and Peking Union Medical College. Ethical approval was granted by the Medical Ethics Committee of Peking Union Medical College (SB2024057). All protocols adhered to the Declaration of Helsinki and the Declaration of the World Medical Association, and informed consent forms were signed by all individuals or their family members.

[0049] Example 1. Decreased TDO2 expression in the liver of elderly individuals 1. Experimental Procedure (1) By analyzing the normal human liver sample transcription sequencing database (GEO database; https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE107039), the population age was divided into two age groups: 20-50 years old and 60-90 years old. The transcriptional levels of TDO2 in these two age groups were compared. The total number of normal human liver samples included was n=33; the specific expression data corresponded to the GEO database subsequence GSE107037.

[0050] (2) Liver tissue samples were collected from individuals aged 24 to 98 years. The number of healthy donor liver tissue samples was n=26. 20 mg of each sample was accurately weighed using an analytical balance and placed in a 1.5 ml EP tube. 200 μL of RIPA lysis buffer containing a protease inhibitor and two 3 mm grinding balls were added to each tube. The tubes were then ground in a pre-cooled low-temperature grinder. After grinding, the tubes were centrifuged at 4°C and 15000 g for 15 min to remove the precipitate. The supernatant was then used for BCA protein quantification. 30 μg of protein was loaded per well, and Western blotting was performed to detect the expression level of TDO2 in each sample.

[0051] 2. Experimental Results Compared to the 20-50 age group, the 60-90 age group had lower liver TDO2 levels. Figure 1A In addition, the protein level of TDO2 gradually decreases with age in older adults. Figure 1B ).

[0052] Example 2. Decreased TDO2 levels in the liver of aged rats 1. Experimental Procedure (1) Four 3-month-old and four 24-month-old C57BL / 6J mice were used to isolate primary hepatocytes from the mice using a non-circulating collagenase perfusion technique. The procedure was briefly as follows: After anesthetizing the mice, a solution containing 0.5% type IV collagenase was perfused via the portal vein. The liver was then removed, gently minced, and filtered through a 70 μm cell sieve. The cell suspension was centrifuged at 50g for 3 minutes at 4°C to precipitate the hepatocytes.

[0053] (2) RNA sequencing was performed by Beijing Annoroad Gene Technology Co., Ltd. The brief steps are as follows: Total RNA was extracted and quality assessed using the Bioanalyzer 2100 system, and sequencing libraries were constructed using the NEBNext® Multiplex Oligos kit. After library standardization and pooling, sequencing was performed on the Illumina NovaSeq X Plus platform (San Diego, USA). Image processing and base identification were performed using Solexa pipeline version 1.8 (offline base identification software, v1.8; San Diego, USA).

[0054] 2. Experimental Results The liver TDO2 level in older mice was significantly lower than that in younger mice, and drug metabolism-related genes were downregulated. Figure 2 ).

[0055] Example 3. TDO2 deficiency leads to increased aging in mice; TDO2 supplementation alleviates liver aging. 1. Experimental Procedure (1) TDO2-deficient mice - / - Background for C57BL / 6J, provided by the research group of Yan Chang and Wei Wei, Anhui Medical University. TDO2 knockout method: AAV8-TBG-shTdo2, with an exemplary target sequence of 5′-GTGAACGACGACTGTCATA-3′ (SEQ ID No. 1), 2×10⁻⁶. 11 vg / animal, administered via tail vein injection.

[0056] (2) Tdo2 - / -Mice were drained of blood via cardiac perfusion as follows: Mice were anesthetized with 1% pentobarbital. The abdominal and thoracic cavities were quickly opened to expose the liver and heart. The right atrial appendage was then cut open, and a needle was inserted into the left ventricle. PBS was used for perfusion until no more blood flowed out, and the liver color lightened, indicating complete drainage. The liver was completely removed, a portion was flash-frozen in liquid nitrogen, and the remaining two intact lobes were fixed with PFA and embedded in OCT. The PFA-fixed samples were then embedded in paraffin, and subsequent paraffin sections were cut for immunohistochemistry and immunofluorescence staining. The OCT-embedded samples were frozen sections and subsequently stained with β-gal and Sirius.

[0057] 2. Experimental Results Comparison of Sirius staining and Oil Red staining showed that TDO2 knockout mice exhibited more severe liver fibrosis and hepatic fat accumulation. Furthermore, β-gal and p16Ink4a staining also demonstrated that TDO2 deficiency exacerbated liver aging. Figure 3A When TDO2 is overexpressed, the level of liver aging decreases ( ). Figure 3B ).

[0058] Example 4. TDO2 knockout mice showed reduced liver detoxification capacity and accumulated more toxins. 1. Experimental Procedure (1) In the exogenous toxin treatment model, C57BL / 6 wild-type (WT) mice and TDO2 knockout mice (knockout method as in Example 3) were treated with trans oleic acid (EA, 50 mg / kg), 2-amino-1-methyl-6-phenylimidazolium[4,5-b]pyridine (PhIP, 5 mg / kg) and acetaminophen (APAP, 50 mg / kg) by oral gavage three times a week for 16 weeks, and liver tissue was collected.

[0059] (2) Following the method described above, mice were perfused with PBS to remove blood from the liver. The liver tissue was homogenized in physiological saline at a mass-to-volume ratio of 1:3, and three volumes of acetonitrile were added to precipitate the protein. The mixture was vortexed and centrifuged twice at 14,000 rpm (5 minutes each time). Finally, 5 μL of the supernatant was taken for analysis by liquid chromatography-mass spectrometry (LC-MS).

[0060] 2. Experimental Results Compared with the control group, TDO2 knockout mice had more drug accumulation in their livers after 16 weeks of continuous treatment with EA, PHIP, and APAP. Figure 4 ).

[0061] Example 5. TDO2 knockout leads to a decrease in the levels of AHR, PXR, CAR and their downstream CYPs. 1. Experimental Procedure As described above, TDO2 knockout mice (knockout method as in Example 3) and wild-type mice were anesthetized, and then the blood in the liver was drained by perfusion of PBS through the heart. Then, 20 mg of liver tissue was cut off, and the tissue was lysed with RIPA lysis buffer containing protease inhibitors. The tissue was ground at low temperature, and then the precipitate was removed by centrifugation. The supernatant was collected to verify the changes in the expression levels of PXR, AHR, CAR and CYPs after TDO2 knockout.

[0062] 2. Experimental Results Immunoblotting experiments showed that TDO2 knockout led to a decrease in AHR, PXR, CAR, and CYPs. Figure 5 ).

[0063] Example 6. TDO2 deficiency leads to increased glycogen accumulation and decreased NADPH. 1. Experimental Procedure (1) Primary hepatocyte isolation and culture method: Primary hepatocytes were isolated from C57BL / 6J mice using a non-circulating collagenase perfusion method. The specific operation steps are as follows: After anesthetizing the mice, a solution containing 0.5% type IV collagenase was perfused through the portal vein. The liver was removed, gently cut into small pieces, and filtered through a 70 μm cell sieve. The filtered cell suspension was placed at 4°C and centrifuged at 50×g for 3 minutes to collect the hepatocyte pellet. The isolated hepatocytes were seeded in William's E medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. The hepatocytes were treated with EA, PHIP, and APAP, respectively, for subsequent detection of glycogen and NADPH.

[0064] (2) Glycogen content was determined using a glycogen assay kit (Abcam) strictly following the manufacturer's experimental protocol. The specific steps were as follows: 20 mg of liver tissue was taken, washed with pre-cooled phosphate-buffered saline (PBS), and resuspended in 500 μL of double-distilled water (ddH2O). The homogenate was homogenized using a homogenizer. The homogenate was boiled for 30 minutes to inactivate enzyme activity, and then centrifuged at 18,000 × g for 10 minutes at 4°C. The supernatant was collected for subsequent testing, and a blank control well was included for each sample. The absorbance was measured at 570 nm using a Synergy™ H1 microplate reader to analyze glycogen content.

[0065] (3) Following the method described above, mouse liver tissue was taken and the NADPH concentration in cells and tissues was detected using the Beyotime kit (product number: S0180S, Beyotime) in accordance with the manufacturer's operating procedures.

[0066] 2. Experimental Results The absence of TDO2 leads to greater glycogen accumulation in hepatocytes. Figure 6A Furthermore, after drug treatment, the NADPH level of hepatocytes in the TDO2 knockout group was lower ( Figure 6B ).

[0067] Example 7. Lactic acid can increase TDO2 levels, reduce liver aging, and decrease ROS levels. 1. Experimental Procedure (1) Prepare drinking water containing lactic acid at a concentration of 6 μM for mice. Administer the solution to the mice continuously for 6 months, and then analyze the liver aging status.

[0068] (2) The mice were drained of blood through cardiac perfusion as follows: After anesthetizing the mice with 1% pentobarbital, the abdominal and thoracic cavities were quickly opened to expose the liver and heart. The right atrial appendage was then cut open with scissors, and a needle was inserted into the left ventricle. PBS was used for perfusion until no more blood flowed out, and the liver color lightened, indicating that the liver blood had been drained. The liver was completely removed, a portion was flash-frozen in liquid nitrogen, and the remaining two intact lobes were fixed with PFA and embedded in OCT. The PFA-fixed liver was then embedded in paraffin, and subsequent paraffin sections were cut for immunohistochemistry and immunofluorescence staining.

[0069] (3) Take mouse liver tissue and use Thermo Fisher Scientific kit (catalog number: A22188) to detect the ROS concentration in cells and tissues according to the manufacturer's operating procedure.

[0070] 2. Experimental Results In this embodiment, a blank vector control group (Vehicle, ordinary drinking water without added lactic acid) and a lactic acid intervention group were set up. Immunohistochemical staining and ROS quantification were performed on mouse liver tissue to detect the proportion of TDO2 positive areas and the proportion of aging markers SA-β-Gal and p16Ink4a positive areas in the liver.

[0071] Immunohistochemical quantitative results showed that the liver TDO2 of control mice was... + The proportion of positive areas was low; after long-term lactic acid intervention, the proportion of TDO2 positive staining areas increased significantly, and the two groups were statistically significant (p<0.0001), confirming that lactic acid can significantly increase the expression level of TDO2 in liver tissue.

[0072] SA-β-Gal and p16Ink4a are classic markers of cellular senescence, and the positive staining area of ​​both can directly characterize the degree of liver aging. (Control group SA-β-Gal) + The region accounted for approximately 27%, and the lactate intervention group had SA-β-Gal + The positive area decreased significantly to approximately 13-14%, with a significant difference between the two groups (p=0.001); the control group p16Ink4a +The area of ​​positive p16Ink4a was approximately 37-38% in the first group. After lactate treatment, the area of ​​positive p16Ink4a decreased significantly to less than 18%, and the difference between the two groups was extremely significant (p < 0.0001). Figure 7A ).

[0073] The above results indicate that long-term lactate intervention can significantly reduce the proportion of senescent cells in the liver and effectively alleviate the liver aging process.

[0074] ROS are key oxidative stress molecules that induce cellular senescence and liver tissue damage. In the control group, the relative ROS level in the liver of mice was above 7.5; in the lactate group, the relative ROS level in the liver tissue was significantly reduced to below 2, with a highly significant difference between the two groups (p < 0.0001). This suggests that lactate can alleviate oxidative stress damage to the liver, reduce ROS accumulation, and inhibit liver aging through the oxidative stress pathway. Figure 7B ).

[0075] In summary, long-term oral administration of lactate can upregulate TDO2 expression, reduce ROS content in liver tissue, and decrease the proportion of senescent positive cells, ultimately improving liver aging and providing a candidate for the development of liver anti-aging drugs.

Claims

1. The use of lactic acid in the preparation of a medicament for improving liver aging in mammals, wherein the medicament is selected from any one or a combination of the following: It upregulates hepatic tryptophan 2,3-dioxygenase (TDO2) expression, reduces hepatic ROS levels, decreases the proportion of hepatic SA-β-Gal positive cells, and decreases the proportion of hepatic p16Ink4a positive cells.

2. The use according to claim 1, wherein: The drug is prepared into an oral or injectable formulation; The oral formulations are selected from: solutions, suspensions, tablets, capsules, granules, powders, enteric-coated formulations, sustained-release formulations, and controlled-release formulations; The injectable formulation is selected from: injection solution, lyophilized powder for injection; The mammal in question is a human.

3. The use of TDO2 detection reagents in the preparation of the kit, including: The kit is used to characterize the degree of liver aging in subjects; The TDO2 detection reagent can quantify the TDO2 expression level in liver tissue samples of subjects; The expression level refers to either the nucleic acid expression level or the protein expression level.

4. The use according to claim 3, wherein: When the expression level is a nucleic acid expression level, the TDO2 detection reagent contains: a TDO2-specific primer or a TDO2-specific probe; When the expression level is a protein expression level, the TDO2 detection reagent contains: a TDO2-specific antibody or its antigen-binding fragment.

5. The use according to claim 3, wherein: If the TDO2 expression level of the first subject or the first subject group is statistically significantly higher than that of the second subject or the second subject group, then the liver aging degree of the first subject or the first subject group is determined to be lower than that of the second subject or the second subject group. Alternatively, if the TDO2 expression level of the first subject or the first subject group is statistically significantly lower than the TDO2 expression level of the second subject or the second subject group, then the liver aging degree of the first subject or the first subject group is determined to be higher than that of the second subject or the second subject group. Alternatively, if the TDO2 expression level of the first subject or the first subject group is statistically significantly higher than the TDO2 expression level of the second subject or the second subject group, then the age of the first subject or the first subject group is determined to be lower than that of the second subject or the second subject group. Alternatively, if the TDO2 expression level of the first subject or the first subject group is statistically significantly lower than the TDO2 expression level of the second subject or the second subject group, then the age of the first subject or the first subject group is determined to be higher than that of the second subject or the second subject group.

6. The use according to claim 3, wherein: When the expression level is a nucleic acid expression level, the kit further comprises any one or a combination of the following: RNA preservation reagent, RNA extraction reagent, reverse transcription reagent, sequencing buffer, PCR amplification reagent, nucleic acid purification reagent, hybridization buffer, sequencing library construction reagent; When the expression level is a protein expression level, the kit further comprises any one or a combination of the following: ELISA coating buffer, ELISA washing buffer, ELISA stop solution, protein loading buffer, protein ladder, electrophoresis buffer, transfer buffer, paraformaldehyde tissue fixation solution, antigen retrieval solution, permeabilization solution, blocking solution, fluorescent secondary antibody, enzyme-labeled secondary antibody, chromogenic substrate, luminescent substrate, and mass spectrometry sample processing reagent.

7. The uses of TDO2 knockout or TDO2 knockdown reagents in the preparation of animal models, including: The animal model is a liver aging model; The animal model is used for drug screening; The animal is a rodent; preferably selected from: mice, rats, rabbits, and guinea pigs; The mice were selected from the following strains: C57BL / 6J, C57BL / 6N, BALB / c, DBA / 2, ICR, and KM.

8. The use according to claim 7, wherein the model presents with symptoms selected from the following: Liver fibrosis, Fat accumulation in the liver Reduced liver function Decreased AHR expression levels in liver tissue The expression level of PXR in liver tissue is decreased. Decreased CAR expression levels in liver tissue Decreased CYP expression levels in liver tissue The expression level of NADPH in liver tissue is decreased. Glycogen accumulation.

9. The use according to claim 7, wherein: When the candidate drug is administered to the animal model, if the expression level or activity of TDO2 in the liver tissue increases compared to before the administration of the candidate drug, the candidate drug is determined to improve liver aging. If the expression level or activity of TDO2 in liver tissue does not change significantly or decreases when the candidate drug is administered to the animal model, then the candidate drug is determined to have limited ability to improve liver aging or liver aging. The expression level refers to either the nucleic acid level or the protein level.

10. The use according to claim 7, wherein: The TDO2 knockout reagent comprises any one or a combination of the following: Cas9 nuclease or Cas9 expression vector, TDO2-specific sgRNA or its expression plasmid, donor template, Cre-loxP element; preferably, the TDO2 knockout reagent comprises at least: Cas9 nuclease or Cas9 expression vector, TDO2-specific sgRNA or its expression plasmid. The TDO2 knockdown reagent comprises any one or a combination of the following: antisense oligonucleotides, siRNA, dsRNA, esiRNA, shRNA, and CRISPRi.

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