Novel aromatic hydrocarbon receptor signaling pathway agonist and application thereof

Abstract

The invention discloses a novel aromatic hydrocarbon receptor signaling pathway agonist and application thereof, and belongs to the technical field of medical preparations. The invention aims to provide a cell model for aromatic hydrocarbon receptor signal channel research and is used for aromatic hydrocarbon receptor signal channel research. The invention discloses the effect of aristololactam I in activation of an AhR signal channel for the first time. The prepared renal tubular epithelial cells with activated aromatic hydrocarbon receptor signal channels can be used for preparing cell models for screening candidate drugs for resisting liver and / or kidney diseases by taking AhR as a target spot. In practical application, AhR in renal tubular epithelial cells with activated aromatic hydrocarbon receptor signal channels can be used as a target spot to screen candidate drugs for resisting liver and / or kidney diseases.

Description

Technical Field

[0001] The present application belongs to the technical field of medical preparations, and specifically relates to a novel aromatic hydrocarbon receptor signaling pathway agonist and its application. Background Art

[0002] The aryl hydrocarbon receptor (AhR) is a highly evolutionarily conserved ligand-activated transcription factor that is widely distributed in various tissues and cells in the human body and regulates a variety of biological and toxicological effects. AhR has been shown to be expressed in a variety of tissues, such as the kidney, liver, and lung, and plays an important role in various physiological and pathological processes. In recent years, research on AhR has become increasingly in-depth, and research on it as a drug target has also increased. AhR can be activated by some endogenous and exogenous compounds: exogenous ligands include polycyclic aromatic hydrocarbons (PAHs), dioxins (TCDD), polychlorinated biphenyls (PCBs), etc.; endogenous ligands of AhR include tryptophan metabolites kynurenine (KYN) and 6-formyl indole [3,2-b] carbazole (FICZ), etc. After binding to AhR, these ligands can mediate toxic effects, regulate cell proliferation, and regulate the immune system. For example, KYN can stimulate cardiomyocyte proliferation and cardiac angiogenesis through AhR; FICZ can promote bone regeneration through AhR, etc. In addition, Wu et al. found in an experiment of bleomycin-induced pulmonary fibrosis in mice that the pulmonary fibrosis of AhR knockout mice was aggravated compared with wild-type mice. These evidences all indicate that AhR is involved in complex and diverse physiological and pathological processes and is an important target.

[0003] AhR is closely related to a variety of liver and kidney diseases. Studies have found that AhR is involved in chemical defense and metabolic regulation of endogenous substances in the liver, and regulates oxidative stress and apoptosis in the kidneys. Kyn plays an important role in the central nervous system and peripheral tissues by activating AhR, including suppressing immune responses and participating in cancer occurrence. FICZ can promote the regeneration and repair of liver cells and reduce liver damage. At the same time, FICZ can also improve kidney function by regulating the metabolic process of kidney cells. In addition, the activity of AhR also affects kidney fibrosis and inflammatory response. Studies have shown that the absence or dysfunction of AhR can lead to liver fatty degeneration, liver fibrosis, liver cancer, glomerulonephritis, tubular damage, etc.

[0004] Aristolochic acid compounds are an important class of natural products that are widely found in traditional Chinese medicine. Previous studies have found that aristolochic acid compounds are significantly toxic and carcinogenic to the liver and kidneys, leading to severe liver and kidney damage, manifested as pathological changes such as hepatocyte necrosis and tubular interstitial fibrosis. Kim et al. found that aristolochic acid exerts carcinogenic and toxic effects in vivo by inducing oxidative stress and DNA damage. Summary of the invention

[0005] The purpose of this application is to provide a cell model for studying the aryl hydrocarbon receptor signaling pathway and to be used for studying the aryl hydrocarbon receptor signaling pathway.

[0006] The present application provides a method for preparing target renal tubular epithelial cells with activated aryl hydrocarbon receptor signaling pathway, the method comprising contacting the renal tubular epithelial cells with an agonist of aryl hydrocarbon receptor (AhR) to obtain the target renal tubular epithelial cells.

[0007] The present application also provides a method for activating an aryl hydrocarbon receptor signaling pathway in renal tubular epithelial cells, the method comprising contacting the renal tubular epithelial cells with an agonist of an aryl hydrocarbon receptor (AhR) to activate the aryl hydrocarbon receptor signaling pathway in the renal tubular epithelial cells.

[0008] The present application also provides a method for upregulating, improving or increasing the expression of AhR downstream genes AhRR and CYP1A1 in renal tubular epithelial cells, the method comprising contacting the renal tubular epithelial cells with an agonist of the aryl hydrocarbon receptor (AhR) to upregulate, improve or increase the expression of AhR downstream genes AhRR and CYP1A1 in the renal tubular epithelial cells.

[0009] Furthermore, in the method described above, the agonist of the aryl hydrocarbon receptor (AhR) is aristolochic acid lactam I.

[0010] Furthermore, in the method, the content of aristolochic acid lactam I in the system for contacting the renal tubular epithelial cells with the agonist of the aryl hydrocarbon receptor (AhR) is 10 -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M or 10 -5 M,

[0011] Furthermore, the amount of aristolochic acid lactam I added is 10 -7 M or 10 -6 M.

[0012] In this application, "AhR" refers to the aryl hydrocarbon receptor, which is a basic helix-loop-basic helix (bHLH) Per-Arnt-Sim homology domain (PAS) protein and belongs to the bHLH superfamily of transcription factors. The aryl hydrocarbon receptor signaling pathway refers to the process in which AhR located in the cytoplasm binds to a ligand that enters the cell, enters the nucleus through the nuclear pore and forms a dimer with the aryl hydrocarbon receptor nuclear translocator (ARNT). The dimer formed can bind to the xenobiotic response element (XRE) in DNA to promote or regulate the intracellular signal transduction process of the expression of downstream genes.

[0013] The aryl hydrocarbon receptor signaling pathway agonist may be a substance that activates the aryl hydrocarbon receptor signaling pathway.

[0014] The agonist may be a substance that activates the aryl hydrocarbon receptor signaling pathway in renal tubular epithelial cells.

[0015] The activation of the aryl hydrocarbon receptor signaling pathway or the activation of the aryl hydrocarbon receptor signaling pathway can be manifested as up-regulation, enhancement or increase in the expression of AhR downstream genes in renal tubular epithelial cells.

[0016] The AhR downstream genes may be AhRR (NCBI Reference Sequence: NM_001377236, updated on 2024-10-29) and CYP1A1 (NCBI Reference Sequence: NM_000499, updated on 2024-10-15).

[0017] The agonist prepared by the aristolochic acid lactam I can be used to prepare renal tubular epithelial cells with activated AhR signaling pathway. Renal tubular epithelial cells with activated AhR signaling pathway can be used to screen new anti-EMT drugs (drugs against renal tubular epithelial-to-mesenchymal transition (EMT)).

[0018] Aryl Hydrocarbon Receptor (AHR) is a transcriptional regulator that can bind to ligands in cells. Under normal circumstances, AHR exists in the cytoplasm and forms complexes with a variety of chaperone proteins. However, after binding to the ligand, AHR enters the nucleus through the nuclear pore and forms a dimer with the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT). The dimer formed can bind to the xenobiotic response element (XRE) in DNA to promote downstream gene expression. AHR can also bind to other transcription factors in the nucleus, thereby affecting the expression of genes regulated by the bound transcription factors.

[0019] Furthermore, in the method, the renal tubular epithelial cells may be isolated cells.

[0020] The above method can be used to prepare renal tubular epithelial cells with activated AhR signaling pathway. Renal tubular epithelial cells with activated AhR signaling pathway can be used to screen new anti-EMT drugs (drugs against renal tubular epithelial-to-mesenchymal transition (EMT)).

[0021] In the present application, the chemical structural formula of the aristolochic acid lactam I is as shown in formula (Ⅰ), and its CAS number is: 13395-02-3 (ChemicalBook: https: / / www.chemicalbook.com / ProductIndex.aspx).

[0022]

[0023] The present application also provides the use of aristolochic acid lactam I in the preparation of an agonist of the aromatic hydrocarbon receptor signaling pathway.

[0024] The present application also provides a cell model, which is a target renal tubular epithelial cell obtained according to a method comprising the following steps: contacting isolated renal tubular epithelial cells with an agonist of the aryl hydrocarbon receptor (AhR) to obtain the target renal tubular epithelial cell, wherein the expression level of the fibrosis marker gene in the target renal tubular epithelial cell is higher than that in the isolated renal tubular epithelial cell.

[0025] Furthermore, in the cell model, the content of aristolochic acid lactam I in the system in which the renal tubular epithelial cells are contacted with an agonist of the aryl hydrocarbon receptor (AhR) is 10 -10 M, 10 -9 M, 10 -8M, 10 -7 M, 10 -6 M or 10 -5 M,

[0026] Furthermore, the content of aristolochic acid lactam I can be 10 -7 M or 10 -6 M.

[0027] In the present application, the system for contacting the renal tubular epithelial cells with the agonist of the aryl hydrocarbon receptor (AhR) may include aristolochic acid lactam I and a culture medium of mammalian cells.

[0028] In the present application, the culture medium of the animal cells is a common culture medium on the market. Including but not limited to: natural culture medium, synthetic culture medium and / or serum-free culture medium. Natural culture medium is mainly taken from animal fluid or separated and extracted from animal tissue. Its advantages are rich nutrients and good culture effect, and its disadvantages are complex ingredients and limited sources. There are many types of natural culture medium / fluid, including biological fluids (such as serum, fetal bovine serum); tissue immersion fluid (such as embryo immersion fluid); coagulant (such as plasma), etc. Synthetic medium (synthetic medium), also known as combined culture medium, is prepared by sequentially adding accurately weighed high-purity chemical reagents and distilled water, and its components (including trace elements) and their amounts are exactly known. Synthetic culture medium is generally used in laboratory research with high quantitative requirements such as nutrition, metabolism, genetics, identification and bioassay. For example: 1640, DMEM. Serum-free culture media and reagents are widely used to culture mammalian and invertebrate cells to prepare monoclonal antibodies, viral antigens and recombinant proteins, etc. Most serum-free culture media contain transferrin, which transports ions into cells, insulin, which regulates glucose uptake, and some proteins, albumin, fibrin, fetuin, etc. These proteins play various functions in cell culture, such as providing the matrix required for cell attachment, resisting the shear force of bioreactors, and acting as carriers of lipids and other growth and differentiation factors. For example: serum-free culture medium for mesenchymal stem cells

[0029] In some embodiments of the present application, the culture medium of the mammalian cells may be a DMEM culture medium containing 10 g / 100 mL fetal bovine serum.

[0030] In some embodiments of the present application, the system may include 10 -10 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0031] In some embodiments of the present application, the system may include 10 -9 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0032] In some embodiments of the present application, the system may include 10 -8 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0033] In some embodiments of the present application, the system may include 10 -7 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0034] In some embodiments of the present application, the system may include 10 -6 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0035] In some embodiments of the present application, the system may include 10 -6 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0036] The present application also provides the use of a composition in any of the following items, wherein the composition comprises aristolochic acid lactam I and a culture medium of mammalian cells,

[0037] A1), use of the composition in preparing renal tubular epithelial cells in which the aryl hydrocarbon receptor signaling pathway is activated;

[0038] A2), use of the composition in activating the aryl hydrocarbon receptor signaling pathway in renal tubular epithelial cells;

[0039] A3) Use of the composition for up-regulating, improving or increasing the expression of AhR downstream genes AhRR and CYP1A1 in renal tubular epithelial cells.

[0040] Furthermore, the renal tubular epithelial cells are in vitro cells.

[0041] In some embodiments of the present application, the composition may contain 10 -10 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0042] In some embodiments of the present application, the composition may contain 10 -9 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0043] In some embodiments of the present application, the composition may contain 10 -8 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0044] In some embodiments of the present application, the composition may contain 10 -7 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0045] In some embodiments of the present application, the composition may contain 10 -6 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0046] In some embodiments of the present application, the composition may contain 10 -6 DMEM medium containing M aristolochic acid lactam I and 10 g / 100 mL fetal bovine serum.

[0047] The beneficial technical effects achieved by this application are as follows:

[0048] This application discloses for the first time the role of aristolochic acid lactone I in activating the AhR signaling pathway.

[0049] The renal tubular epithelial cells with activated aryl hydrocarbon receptor signaling pathway prepared in the present application can be used to prepare a cell model for screening candidate drugs against liver and / or kidney diseases with AhR as a target. In practical applications, AhR in renal tubular epithelial cells with activated aryl hydrocarbon receptor signaling pathway can be used as a target to screen candidate drugs against liver and / or kidney diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 These are the results of the direct activation effect of aristolochic acid lactone I on the AhR signaling pathway of mouse liver cancer CBG2.8D cells.

[0051] Figure 2 These are the test results of the toxic effects of aristolochic acid lactam I on renal tubular epithelial cells.

[0052] Figure 3 These are the results of the detection of the expression of downstream genes of the AhR signaling pathway in human renal tubular epithelial HKC cells induced by aristolochic acid I.

[0053] Figure 4 This is the verification result that downregulation of AhR expression inhibits the induction effect of aristolochic acid I on genes downstream of the AhR signaling pathway in human renal tubular epithelial HKC cells. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0055] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0056] In the following embodiments, the HKC cells were purchased from the Cell Resource Center of the Chinese Academy of Medical Sciences, and the cell line number is 1101HUM-PUMC00016, hereinafter referred to as HKC cells. CBG2.8 cells were purchased from Tuogan (Shenzhen) Technology Creative Co., Ltd., and the cell line number is SLA0101. The CCK8 kit is a product of Sangon Biotech (Shanghai), and the item number is E606335. TCDD is a product of Wellington Laboratory, and the item number is 513CS6. . DMSO is a product of Sigma-Aldrich, and the item number is D8418. Aristolochic acid lactam I is a product of Dester Biotechnology Co., Ltd. (Chengdu), and its item number is DM0055. Human AhR interfering RNA (AhR siRNA) and non-targeted siRNA (NC) were designed and synthesized by SyngenTech (Beijing). Lipofectamine RNAiMAX kit is a product of Invitrogen (USA), and its item number is: 13778150. Opti-MEM TM Reduced Serum Medium is a product of Gibco (USA), with a catalog number of 51985034. GeneJET RNA purification kit is a product of Thermo Fisher Scientific (USA), with a catalog number of K0732. RevertAid FirstStrand cDNA Synthesis kit is a product of Thermo Fisher Scientific (USA), with a catalog number of K1622. qPCR Master Mix is ​​a product of Promega (USA), with the catalog number A6001. Luciferase Assay System kit is a product of Promega (USA), with the catalog number E1501. Luciferase Cell Culture Lysis 5X Reagent kit is a product of Promega (USA), with the catalog number E1531. Primers were synthesized by Sangon Biotech (Shanghai). NanoDrop spectrophotometer: 2000 / 2000c is a product of Thermo (USA). Real-time fluorescence quantitative PCR instrument: QuantStudion TM6 Flex Real-Time PCR System is a product of Thermo (USA). ELISA reader: Tecan 2000pro is a product of Tecan (Switzerland). Chemiluminescence detector: -Multi+Detection System is a product of Promega.

[0057] In the following embodiments, the chemical structural formula of aristolochic acid lactam I is as shown in formula (Ⅰ), and its CAS number is: 13395-02-3 (ChemicalBook: https: / / www.chemicalbook.com / ProductIndex.aspx).

[0058]

[0059] In the present application, the structural formula of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is as shown in formula (II),

[0060]

[0061] Unless otherwise specified, the medium used for cell culture in the following examples is a low serum medium, which is a DMEM medium containing 10 g / 100 mL of fetal bovine serum.

[0062] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0063] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. The t-test was used for the test, and P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) or P < 0.001 (***) indicated a very significant difference.

[0064] The following examples show that aristolochic acid lactone I can activate the AhR signaling pathway in both liver and kidney cells, especially after the addition of AhR interfering RNA (AhR siRNA) in renal tubular epithelial cells, the expression of the above genes is inhibited. This discovery provides a new direction and theoretical basis for the development of new anti-liver and kidney disease drugs with AhR signaling pathway function regulation.

[0065] Example 1. Direct activation of AhR signaling pathway in mouse liver cancer CBG2.8D cells by aristolochic acid lactam I

[0066] CBG2.8D luciferase reporter gene assay cells were cultured at 2×10 4Inoculate cells at a concentration of 100 μL / mL into a 96-well plate, with 100 μL of cell suspension per well. Culture overnight and wait for cells to adhere to the wall. Take out the 96-well plate and aspirate the original culture medium. Set up a blank group, a control group, and a drug-treated group for the experiment. Treat them as follows:

[0067] Blank group: 100 μL / well of low serum medium was added to the 96-well plate without cells;

[0068] Control group: 100 μL / well of low-serum medium containing 1% dimethyl sulfoxide (DMSO) was added to the 96-well plate containing adherent cells;

[0069] Drug treatment group: 10 -10 M, 10 -7 M, 10 -6 M or 10 -5 M aristolochic acid lactam I low-serum medium 100 μL / well;

[0070] The low serum culture medium is a DMEM culture medium containing 10 g / 100 mL of fetal bovine serum.

[0071] Place the 96-well plate in the incubator and continue to culture for 24 hours. After reaching the end time of the culture, pour out the culture medium in the 96-well plate and wash it once with phosphate buffered saline (PBS). After wiping the 96-well plate dry, stick a light-shielding film on the bottom of the plate, and then add 100 μL of 1× cell lysis buffer to each well and shake for 15 minutes according to the instructions of the Luciferase Assay System kit (purchased from Promega, catalog number: E1501). After the shaking, add 100 μL of luciferase detection reagent to each well and read the fluorescence value with a chemiluminescence detector. This experiment was repeated three times independently, and each aristolochic acid lactam I addition concentration in the blank group, control group and drug treatment group in each independent repeated experiment was set up three times, and the results were averaged.

[0072] The calculation formula is as follows:

[0073] Luciferase relative activity = (OD 药物处理组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )

[0074] The luciferase reporter gene-based bioassay is designed to utilize the biological process that dioxin compounds act on AhR in cells to activate downstream signaling pathways and induce the expression of reporter genes containing dioxin response element (DRE) promoter regulatory sequences in the cell nucleus. Dioxin compounds can induce the expression of luciferase genes in the test cells in a dose-dependent manner, that is, the expression of the reporter gene shows a specific corresponding relationship with the content of dioxin compounds under certain conditions, so that the toxic equivalent of the exposed dioxin compounds relative to the standard compounds can be determined by detecting the reporter gene.

[0075] like Figure 1 As shown, using 10 -7 -10 -5 After CBG2.8D cells were treated with M aristolochic acid I and the positive control TCDD for 24 hours, the relative activity of luciferase in the cells was significantly increased compared with the control group, indicating that 10 -6 M and 10 -5 Aristolochic acid lactam I can effectively activate the AhR signaling pathway in CBG2.8D cells.

[0076] Example 2. Study on the toxic effects of aristolochic acid lactam I on renal tubular epithelial cells

[0077] HKC cells were cultured at 1×10 4 Inoculate cells at a concentration of 100 μL / mL into a 96-well plate, with 100 μL of cell suspension per well. Incubate overnight and wait for cells to adhere to the wall. Take out the 96-well plate, aspirate the original culture medium, and wash once with phosphate buffered saline (PBS). The experiment set up a blank group, a control group, and a drug-treated group. The treatments were as follows:

[0078] Blank group: 100 μL / well of low serum medium was added to the 96-well plate without cells;

[0079] Control group: 100 μL / well of low-serum medium containing 0.1% dimethyl sulfoxide (DMSO) was added to the 96-well plate containing adherent cells;

[0080] Drug treatment group: 10 -8 M, 10 -7 M, 10 -6 M or 10 - 5 M aristolochic acid lactam I low-serum medium 100 μL / well;

[0081] The 96-well plate was placed in an incubator and continued to be cultured for 24 hours or 48 hours. After reaching the end time of the culture, 10 μL of CCK8 reagent was added to each well according to the instructions of the CCK8 kit, and the 96-well plate was placed in the incubator again for incubation for 2 hours, and the absorbance was read with a microplate reader. This experiment was repeated three times for 24 hours or 48 hours, and three replicates were set for each aristolochic acid lactam I addition concentration of the blank group, control group and drug treatment group in each independent repeated experiment, and the results were averaged.

[0082] The calculation formula is as follows:

[0083] Cell viability = (OD 药物处理组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )

[0084] The results are as follows Figure 2 As shown, using 10 -8 -10 -5 HKC cells were treated with M aristolochic acid I for 48 hours, of which 10 -8 M aristolochic acid lactam I had a slight effect on cell proliferation and viability at 48 hours, but the effect was small and non-cytotoxic, so it can be used in subsequent experiments.

[0085] Example 3. Aristolochic acid lactone I induces the expression of downstream genes of AhR signaling pathway in human renal tubular epithelial HKC cells

[0086] HKC cells were cultured at 2×10 5 The cells were inoculated into a 6-well plate at a concentration of 100 / mL, with 2 mL of cell suspension in each well. Incubate overnight and wait for the cells to adhere to the wall. Then remove the 6-well plate, aspirate the original culture medium, and wash once with PBS. The experiment was divided into a control group, an aristolochic acid lactone I treatment group, and a TCDD treatment group. Three replicates were set for each added concentration of the control group, the aristolochic acid lactone I treatment group, and the TCDD treatment group.

[0087] Control group: 2 mL / well of low-serum medium containing 0.1% dimethyl sulfoxide (DMSO) was added to the 6-well plate containing adherent cells;

[0088] Aristolochic acid lactam I treatment group: 10 -7 M, 10 -6 2 mL / well of low-serum medium containing M aristolochic acid lactam I;

[0089] 10 -10 M TCDD treatment group: 10 -8 M TCDD low serum medium 2 mL / well;

[0090] The low serum culture medium is a DMEM culture medium containing 10 g / 100 mL of fetal bovine serum.

[0091] After culturing the cells for 48 hours, total cell RNA was extracted to detect the relative expression levels of AhRR (NCBI Reference Sequence: NM_001377236, updated: 2024-10-29) and CYP1A1 (NCBI Reference Sequence: NM_000499, updated: 2024-10-15). Total RNA was extracted using the GeneJET RNA purification kit. The quality and concentration of RNA were analyzed using a NanoDrop2000 / 2000c spectrophotometer (Thermo Fisher Scientific). Using 1 μg of total RNA as raw material, cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit. Use qPCR Master Mix was used to perform real-time fluorescence quantitative analysis on 20-fold diluted cDNA samples. The thermal cycle conditions were as follows: 95°C for 2 min, denaturation at 95°C for 40 times, each time for 15 s, annealing at 60°C for 20 s, and extension at 72°C for 20 s. The results were analyzed by 2 -ΔΔCT The value method was used for analysis. The expression of glyceraldehyde phosphate dehydrogenase (GAPDH) was not affected by aristolochic acid I and could be used as an internal reference. The experiment ensured that the amplification efficiency of the primers used was 95-105%. Each sample was tested three times and the results were averaged. Primers were designed using Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ), and the sequences were obtained from GenBank (http: / / www.ncbi.nlm.nih.gov / genbank / ). The primer sequences are as follows:

[0092] AhRR-F: 5'-AGGTTTGGTTGGCAGGACT-3' (SEQ ID NO: 1);

[0093] AhRR-R: 5'-GCTCAGATGGTTGGCTGTTC-3' (SEQ ID NO: 2);

[0094] CYP1A1-F: 5'-TCGGCCACGGAGTTTCTC-3' (SEQ ID NO: 3);

[0095] CYP1A1-R: 5'-GGTCAGCATGTGCCCAATCA-3' (SEQ ID NO: 4);

[0096] GAPDH-F: 5'-CATGAGAAGTATGACAACAGCCT-3' (SEQ ID NO: 5);

[0097] GAPDH-R: 5'-AGTCCTTCCACGATACCAAAGT-3' (SEQ ID NO: 6).

[0098] We evaluated the activation of the AhR signaling pathway by detecting the expression of AhR downstream genes AhRR and CYP1A1. Figure 3 As shown in the results, aristolochic acid lactone I can increase the expression of AhRR and CYP1A1 in a time-dependent manner. -7 M and 10 -6 After 48 hours, the expression levels of AhRR in cells treated with M aristolochic acid I were 1.43 and 1.75 times higher than those in the control group, and the expression levels of CYP1A1 were 1.76 and 2.60 times higher than those in the control group. The results showed that aristolochic acid I can stably and effectively activate the AhR signaling pathway in HKC cells. This experiment was repeated three times independently, and each group in each independent repeated experiment was repeated three times.

[0099] Example 4. Down-regulation of AhR expression inhibits the induction of aristolochic acid lactam I on AhR signaling pathway downstream genes in human renal tubular epithelial HKC cells

[0100] HKC cells were cultured at 2×10 5 Inoculate the cells at a concentration of 100 / mL into a 6-well plate, with 2 mL of cell suspension per well. Incubate overnight and wait for the cells to adhere. Take out the 6-well plate and transfect AhR siRNA or non-targeted siRNA (negative control NC). Follow the instructions and use RNAi MAX transfection reagent for transfection. Transfect each siRNA in 3 replicates. After 24 hours of transfection, aspirate the original culture medium and continue to treat according to the following groups:

[0101] Control group: 2 mL / well of low-serum medium containing 0.1% dimethyl sulfoxide (DMSO) was added to the 6-well plate;

[0102] Aristolochic acid lactam I treatment group: 10 -6 2 mL / well of low-serum medium containing M aristolochic acid lactam I;

[0103] The culture was continued for 24 hours, and then the total RNA of the cells was extracted for subsequent research. The low serum culture medium was a DMEM culture medium containing 10 g / 100 mL of fetal bovine serum. The steps of total RNA extraction and real-time fluorescence quantitative analysis were the same as those in Example 3. The siRNA sequence is as follows:

[0104] NC sense strand: 5′-UUC UCC GAA CGU GUC ACG UTT-3′ (SEQ ID NO: 7);

[0105] NC antisense strand: 5′-ACG UGA CAC GUU CGG AGA ATT-3′ (SEQ ID NO: 8);

[0106] AhR siRNA sense strand: 5′-GAA CUU ACA AGA AGG AGA ATT-3′ (SEQ ID NO: 9);

[0107] AhR siRNA antisense strand: 5′-UUC UCC UUC UUG UAA GUU CTT-3′ (SEQ ID NO: 10);

[0108] like Figure 4 As shown, after treatment with AhR siRNA for 48 hours, 10 -6 The expression changes of AhRR and CYP1A1, the downstream genes of the AhR pathway induced by M aristolochic acid I, were reversed, indicating that AhR is the target of aristolochic acid I. This experiment was repeated three times independently, and each group in each independent repeated experiment had three replicates.

[0109] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for preparing target renal tubular epithelial cells with activated aryl hydrocarbon receptor signaling pathway, characterized in that: The method comprises contacting the renal tubular epithelial cells with an agonist of aryl hydrocarbon receptor to obtain target renal tubular epithelial cells.

2. A method for activating the aryl hydrocarbon receptor signaling pathway in renal tubular epithelial cells, characterized in that: The method comprises contacting the renal tubular epithelial cells with an agonist of the aryl hydrocarbon receptor to activate the aryl hydrocarbon receptor signaling pathway in the renal tubular epithelial cells.

3. A method for up-regulating, improving or increasing the expression of AhR downstream genes AhRR and CYP1A1 in renal tubular epithelial cells, characterized in that: The method comprises contacting the renal tubular epithelial cells with an agonist of the aryl hydrocarbon receptor to upregulate, improve or increase the expression of AhR downstream genes AhRR and CYP1A1 in the renal tubular epithelial cells.

4. The method according to any one of claims 1 to 3, characterized in that The agonist of the aromatic hydrocarbon receptor is aristolochic acid lactam I.

5. The method according to claim 4, characterized in that The content of aristolochic acid lactam I in the system for contacting the renal tubular epithelial cells with the agonist of the aromatic hydrocarbon receptor is 10 -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M or 10 - 5 M, Furthermore, the amount of aristolochic acid lactam I added is 10 -7 M or 10 -6 M.

6. Application of aristolochic acid lactone I in the preparation of agonists of the aromatic hydrocarbon receptor signaling pathway.

7. A cell model, characterized in that The cell model is a target renal tubular epithelial cell obtained by a method comprising the following steps: contacting isolated renal tubular epithelial cells with an agonist of an aromatic hydrocarbon receptor to obtain the target renal tubular epithelial cell, wherein the expression level of the fibrosis marker gene in the target renal tubular epithelial cell is higher than that in the isolated renal tubular epithelial cell.

8. The cell model according to claim 7, characterized in that In the cell model, the content of aristolochic acid lactam I in the system in which the renal tubular epithelial cells are contacted with an agonist of the aromatic hydrocarbon receptor is 10 -10 M, 10 -9 M, 10 - 8 M, 10 -7 M, 10 -6 M or 10 -5 M, Furthermore, the content of aristolochic acid lactam I can be 10 -7 M or 10 -6 M.

9. Use of a composition in any of the following, wherein the composition comprises aristolochic acid lactam I and a culture medium of mammalian cells, A1), use of the composition in preparing renal tubular epithelial cells in which the aryl hydrocarbon receptor signaling pathway is activated; A2), use of the composition in activating the aryl hydrocarbon receptor signaling pathway in renal tubular epithelial cells; A3) Use of the composition for up-regulating, improving or increasing the expression of AhR downstream genes AhRR and CYP1A1 in renal tubular epithelial cells.

10. The use according to claim 9, characterized in that: The renal tubular epithelial cells are isolated cells.

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