Novel regulation and control method for renal tubular epithelium mesenchymal transformation

Abstract

The invention belongs to the technical field of microorganisms, particularly relates to animal cells, and more particularly relates to a novel renal tubular epithelium mesenchymal transformation regulation and control method. The invention aims to solve the technical problems of how to research the mechanism of renal tubular epithelium mesenchymal transformation and how to obtain the cell model for researching the mechanism of renal tubular epithelium mesenchymal transformation. In order to solve the technical problem, the invention provides a method for regulating and controlling the expression of a fibrosis marker gene in renal tubular epithelial cells and a corresponding composition. The method provided by the invention can be used for preparing the renal tubular epithelial cells with mesenchymal cell characteristics. The renal tubular epithelial cells with the mesenchymal cell characteristics can be used as a cell model for screening new drugs for resisting renal tubular epithelial mesenchymal transformation. The method has a positive effect on mechanism research and clinical treatment of renal tubular epithelium mesenchymal transformation.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to animal cells. More specifically, the present application relates to a novel method for regulating renal tubular epithelial-mesenchymal transition. Background Art

[0002] Epithelial-to-mesenchymal transition (EMT) is a key pathological process in the progression of chronic kidney disease (CKD), an important mechanism of renal interstitial fibrosis (RIF), and one of the main causes of renal function decline and ultimately end-stage renal disease. Studies have shown that during the EMT process, renal tubular epithelial cells lose their epithelial characteristics and transform into myofibroblasts with mesenchymal cell characteristics. These cells can produce and secrete a large amount of extracellular matrix (ECM), which eventually leads to the occurrence of renal interstitial fibrosis. This process is closely related to the pathological progression of kidney disease. Blocking the role of EMT in renal fibrosis has been shown to significantly reduce fibrotic lesions, indicating that EMT is of great significance in renal interstitial fibrosis. Therefore, regulation of EMT has become an important strategy for the treatment of CKD and renal interstitial fibrosis. Drug development targeting EMT provides new ideas for the treatment of CKD. For example, some drugs can block the occurrence of EMT by inhibiting key signaling pathways in the EMT process (such as TGF-β / Smad, Wnt / β-catenin, etc.). In addition, some traditional Chinese medicines have also shown intervention effects on the EMT process, such as astragalus and cordyceps, thus providing a potential pharmacological approach for the treatment of renal fibrosis.

[0003] The aryl hydrocarbon receptor (AhR) plays a complex regulatory role in the fibrosis of the heart, liver, and lung. − / −Mice were more likely to develop left ventricular hypertrophy in Ang II infusion-induced cardiac fibrosis, and c-Jun / HIF signaling activity and expression levels of the downstream target vascular endothelial growth factor were significantly enhanced compared with wild-type mice. These results suggest that AhR has a pathogenic role in the development of cardiac fibrosis. However, some studies have also shown that the regulation of AhR activity shows a complex mechanism of action. The activation or inhibition of AhR has a dual role in liver and lung fibrosis, which may promote or inhibit this process. Yan et al. found that AhR can inhibit the activation of hepatic stellate cells and the expression of genes required for the occurrence of liver fibrosis, which provides stronger evidence for the negative regulation of AhR on liver fibrosis. However, some people have suggested that AhR may cause liver fibrosis. In wild-type mice, treatment with TCDD or other AhR ligands induced the hepatic expression of fibrotic markers (such as COL1A1 and α-SMA), while in AhR − / − This was not observed in mice. In addition, studies have analyzed AhR expression levels in non-small cell lung cancer cell lines and lung cancer tissues, confirming the negative correlation between AhR protein levels and EMT, and static AhR protein may act as an EMT inhibitor through non-genomic pathways. These results indicate that AhR is associated with fibrosis and EMT.

[0004] Dioxin compounds are exogenous agonists of AhR, among which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has the strongest activation effect on AhR. CH223191 is an effective and specific AhR antagonist, preferentially antagonizing halogenated aromatic AhR agonists such as TCDD. These drugs are widely used in experiments to regulate the activity of AhR signaling pathways, providing tools for studying AhR-based renal tubular epithelial EMT regulation methods.

[0005] In the following examples, by using regulators of AhR signaling pathway activity, we detected a series of renal tubular epithelial-mesenchymal transition markers to evaluate the intensity changes of the EMT effect, and found that TCDD or CH223191 can activate or inhibit the AhR signaling pathway in renal tubular epithelial cells HKC cells, while regulating the expression of some specific EMT markers, which are considered to be key factors in the pathological process of EMT in many literatures. Therefore, the present invention realizes the regulation of the expression of specific markers that reflect the intensity of the EMT effect by regulating the activity of the AhR signaling pathway, provides a new method for regulating renal tubular epithelial-mesenchymal transition, and provides new ideas and strategies for the development of new anti-EMT drugs. Summary of the invention

[0006] The technical problem to be solved by the present application is: how to study the mechanism of renal tubular epithelial-mesenchymal transition and how to obtain a cell model for studying the mechanism of renal tubular epithelial-mesenchymal transition.

[0007] To solve the above technical problems, the present application provides a method for upregulating, improving or increasing the expression of fibrosis marker genes in renal tubular epithelial cells, the method comprising contacting isolated renal tubular epithelial cells with an agonist of the aryl hydrocarbon receptor (AhR) to upregulate, improve or increase the expression of fibrosis marker genes in the renal tubular epithelial cells.

[0008] The present application also provides a method for preparing target renal tubular epithelial cells with upregulated, increased or enhanced expression of fibrosis marker genes, the method comprising contacting isolated renal tubular epithelial cells with an agonist of aryl hydrocarbon receptor (AhR) to obtain the target renal tubular epithelial cells.

[0009] In the present application, the fibrosis marker gene may also be referred to as an epithelial-mesenchymal transition marker gene.

[0010] The expression level of the fibrosis marker gene in the target renal tubular epithelial cells in which the expression of the fibrosis marker gene is upregulated, improved or increased is higher than that in the isolated renal tubular epithelial cells.

[0011] Furthermore, the fibrosis marker genes in the renal tubular epithelial cells include but are not limited to Fibronectin (NCBI Reference Sequence: NM_001365522.2, updated: 2024-11-12), MMP9 (NCBI Reference Sequence: NM_004994.3, updated: 2024-10-29) and IL-6 (NCBI Reference Sequence: NM_001318095.2, updated: 2024-11-12).

[0012] Furthermore, in the method, the agonist of the aryl hydrocarbon receptor (AhR) is dioxin.

[0013] Furthermore, the dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

[0014] Furthermore, in the method, the content of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the system in which the renal tubular epithelial cells are contacted with an agonist of an aryl hydrocarbon receptor (AhR) is 10 -10 M, 10 -9 M, 10 -8 M or 10 -7 M.

[0015] Furthermore, the content of 2,3,7,8-tetrachlorodibenzo-p-dioxin is 10 -8 M.

[0016] The method can be used to prepare renal tubular epithelial cells with mesenchymal cell characteristics. The renal tubular epithelial cells with mesenchymal cell characteristics can be used to screen new anti-EMT drugs (drugs against renal tubular epithelial-to-mesenchymal transition (EMT)).

[0017] The renal tubular epithelial cells with mesenchymal cell characteristics may be target renal tubular epithelial cells in which the expression of the above-mentioned fibrosis marker genes is upregulated, increased or enhanced.

[0018] In this field, dioxin is actually an abbreviation for dioxins. It does not refer to a single substance, but two major categories of organic compounds with similar structures and properties, including many homologues or isomers. Dioxins include 210 compounds. These substances are very stable, have a high melting point, are extremely difficult to dissolve in water, but can be dissolved in most organic solvents. They are colorless and odorless fat-soluble substances, so they are very easy to accumulate in organisms and are seriously harmful to the human body.

[0019] The full names of dioxins are polychlorinated dibenzo-p-dioxin (PCDDs) and polychlorinated dibenzofuran (PCDFs). PCDDs are formed by two oxygen atoms connecting two benzene rings substituted by chlorine atoms; PCDFs are formed by one oxygen atom connecting two benzene rings substituted by chlorine atoms. Each benzene ring can be replaced by 1 to 4 chlorine atoms, thus forming numerous isomers, of which PCDDs have 75 isomers and PCDFs have 135 isomers.

[0020] 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an isomer of polychlorinated dibenzo-p-dioxins (PCDDs).

[0021] The present application also provides a cell model, wherein the cell model 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.

[0022] Furthermore, in the method for preparing the cell model, the agonist of the aryl hydrocarbon receptor (AhR) is a dioxin compound. Furthermore, the dioxin compound is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

[0023] Furthermore, in the method for preparing the cell model, the content of 2,3,7,8-tetrachlorodibenzo-p-dioxin 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 -8 M or 10 -7 M, Furthermore, the content of 2,3,7,8-tetrachlorodibenzo-p-dioxin is 10 -8 M.

[0024] In the present application, the system for contacting the renal tubular epithelial cells with the agonist of the aryl hydrocarbon receptor (AhR) may be an animal cell culture medium containing 2,3,7,8-tetrachlorodibenzo-p-dioxin.

[0025] 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 medium 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 In some embodiments of the present application, the culture medium of the mammalian cells is DMEM culture medium containing 10 g / 100 mL fetal bovine serum.

[0026] The present application also provides a use of a substance, wherein the substance is a substance that regulates the activity of the AhR signaling pathway, and the use is the use of the substance in preparing a drug that regulates the process of renal tubular epithelial cells to epithelial-mesenchymal transition.

[0027] The regulation of the renal tubular epithelial cell to epithelial-mesenchymal transition process may specifically be up-regulating, improving or increasing the expression of fibrosis marker genes in the renal tubular epithelial cells.

[0028] The regulation of the renal tubular epithelial cell to epithelial-mesenchymal transition process may specifically be down-regulation, reduction or decrease of the expression of fibrosis marker genes in the renal tubular epithelial cells.

[0029] Furthermore, in the use described above, the substance is an agonist of the aryl hydrocarbon receptor and / or an antagonist of the aryl hydrocarbon receptor.

[0030] Furthermore, the aryl hydrocarbon receptor agonist is (selected from) dioxin, Further, the dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); Furthermore, the antagonist of aryl hydrocarbon receptor is CH223191.

[0031] Furthermore, in the use described above, the effective dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin is 10 - 10 M, 10 -9 M, 10 -8 M or 10 -7 M.

[0032] Furthermore, the effective dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin is 10 -8 M.

[0033] Furthermore, the effective dose of CH223191 is 10 -5 M.

[0034] Furthermore, in the use described above, the renal tubular epithelial cells are in vitro cells.

[0035] The 2,3,7,8-tetrachlorodibenzo-p-dioxin can be used to prepare a drug for up-regulating, improving or increasing the expression of fibrosis marker genes in renal tubular epithelial cells.

[0036] The CH223191 can be used to prepare a drug for down-regulating, lowering or reducing the expression of fibrosis marker genes in renal tubular epithelial cells.

[0037] The drug can be used for studying the mechanism of tubular epithelial-mesenchymal transition of renal tubular epithelial cells and / or renal tubular epithelial cell fibrosis.

[0038] In the present application, the structural formula of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is as shown in formula (I),

[0039] In this application, the Chinese name of CH223191 is: 1-methyl-N-[2-METHYL-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide, its CAS number is: 301326-22-7 (ChemicalBook: https: / / www.chemicalbook.com / ProductIndex.aspx), and its structural formula is as shown in formula (II):

[0040] The beneficial technical effects achieved by this application are as follows: The present application provides a method for regulating the expression of fibrosis marker genes in renal tubular epithelial cells and a corresponding composition. The method provided in the present application can be used to prepare renal tubular epithelial cells with mesenchymal cell characteristics. The renal tubular epithelial cells with mesenchymal cell characteristics can be used as a cell model to screen new anti-EMT drugs (anti-epithelial-to-mesenchymal transition (EMT) drugs). It has a positive effect on the mechanism research and clinical treatment of renal tubular epithelial-to-mesenchymal transition. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The results show the toxic effect of TCDD on human renal tubular epithelial cells (HKC).

[0042] Figure 2 The results show that TCDD inhibits the expression of aryl hydrocarbon receptor (AhR) in human renal tubular epithelial cells (HKC).

[0043] Figure 3 The results show the regulation of TCDD on the activity of AhR signaling pathway in human renal tubular epithelial cells (HKC).

[0044] Figure 4 The results of TCDD-induced fibrosis in human renal tubular epithelial cells (HKC) were detected by the expression of fibrosis marker genes Fibronectin, MMP9 and IL-6.

[0045] Figure 5 These are the results of CH223191 regulating the expression of fibrosis marker genes Fibronectin, MMP9 and IL-6 in human renal tubular epithelial cells (HKC). DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] In the following examples, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a product of Wellington Laboratory, with a product number of 513CS6. The structural formula of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is as follows:

[0049] In the following examples, the source of CH223191 is a product of Sigma-Aldrich, with the product number 301326-22-7. Its Chinese name is: 1-methyl-N-[2-METHYL-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide, its CAS number is: 301326-22-7 (ChemicalBook: https: / / www.chemicalbook.com / ProductIndex.aspx), and its structural formula is as follows:

[0050] In the following implementation, HKC cells (human renal tubular epithelial cells) were purchased from the Cell Resource Center of the Chinese Academy of Medical Sciences, and the cell line number was 1101HUM-PUMC00016, hereinafter referred to as HKC cells. CCK8 kit is a product of Sangon Biotech (Shanghai), and the product number is E606335. DMSO is a product of Sigma-Aldrich, and the product number is D8418. Lipofectamine RNAiMAX kit is a product of Invitrogen (USA), and the product number is: 13778150. GeneJET RNA purification kit is a product of Thermo Fisher Scientific (USA), and the product number is: K0732. RevertAid First Strand cDNA Synthesis kit is a product of Thermo Fisher Scientific (USA), and the product number is: K1622. GoTaq®qPCR Master Mix is ​​a product of Promega (USA), and the product number is: A6001. Primers were synthesized by Sangon Biotech (Shanghai). NanoDrop spectrophotometer: 2000 / 2000c is a product of Thermo (USA). Real-time fluorescence quantitative PCR instrument: QuantStudion TM 6 Flex Real-Time PCR System is a product of Thermo (USA). Microplate reader: Tecan 2000pro is a product of Tecan (Switzerland).

[0051] 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.

[0052] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0053] 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 < 0.001 (***) indicated a very significant difference.

[0054] Example 1. Study on the toxic effect of TCDD on human renal tubular epithelial cells (HKC) HKC cells were cultured at 1×10 4Inoculate 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. Take out the 96-well plate, aspirate the original culture medium, wash once with phosphate buffered saline (PBS), and obtain a 96-well plate containing adherent cells for subsequent experiments. The experiment set up a blank group, a control group, and a drug-treated group. The treatments were as follows: Blank group: 100 μL / well of low serum medium was added to the 96-well plate without cells; 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; Drug treatment group: 10 -10 M, 10 -9 M, 10 -8 M or 10 -7 100 μL / well of M TCDD low-serum medium.

[0055] Then the 96-well plate was placed in the incubator for further culture for 24 hours or 48 hours (i.e., 24-hour or 48-hour assay group). After reaching the end point of culture (24 hours or 48 hours), 10 μL CCK8 reagent was added to each well according to the instructions of the CCK8 kit, and the 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 each TCDD addition concentration in the blank group, control group, and drug treatment group in each independent repeated experiment was set to 6 replicates, and the results were averaged.

[0056] The calculation formula is as follows:

[0057] The results are as follows Figure 1 As shown, using 10 -10 -10 -7 HKC cells were treated with M TCDD for 48 hours, 10 -9 Up to 10 -7 Although M TCDD has a slight effect on cell viability and proliferation, the effect is small and non-cytotoxic, so it can be used in subsequent experiments.

[0058] Example 2. Expression of aryl hydrocarbon receptor (AhR) in human renal tubular epithelial cells (HKC) HKC cells were cultured at 2×10 5 The cells were inoculated into a 6-well plate at a concentration of 100 cells / mL, with 2 mL of cell suspension per well. The plates were cultured overnight to allow the cells to adhere to the wall. The 6-well plates were then removed, the original culture medium was aspirated, and the plates were washed once with PBS. The experiment was divided into a control group and 10 -8MTCDD treatment group, each treatment group was set up with three replicates.

[0059] 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; 10 -8 M TCDD treatment group: 10 -8 2 mL / well of low-serum medium containing M TCDD.

[0060] After culturing the cells for 48 hours, total cell RNA was extracted to detect the relative expression of AhR (NCBI Reference Sequence: NM_001621.5, updated on 2024-11-12). Total RNA was extracted using the GeneJET RNA purification kit. The quality and concentration of RNA were analyzed using a NanoDrop 2000 / 2000c spectrophotometer (Thermo Fisher Scientific). Using 1 μg of total RNA as the raw material, cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit. The 20-fold diluted cDNA samples were subjected to real-time fluorescence quantitative analysis using GoTaq® qPCR Master Mix. The thermal cycling conditions were as follows: 95°C for 2 min, denaturation at 95°C for 40 times, each 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 expression of glyceraldehyde phosphate dehydrogenase (GAPDH) was not affected by TCDD 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. 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: AhR-F: 5'-CAACAGCAACAGTCCTTGGC-3' (SEQ ID NO: 1); AhR-R: 5'-GTTGCTGTGGCTCCACT-3' (SEQ ID NO: 2).

[0061] We detected the expression of AhR in HKC after adding TCDD. Figure 2 As shown, TCDD can reduce the expression of AhR. -8After 48 hours of M TCDD treatment, the expression level of AhR decreased by 31.18% compared with the control group. The above results show that AhR is expressed in human renal tubular epithelial cells (HKC), and TCDD can inhibit the expression of AhR in HKC. This experiment was repeated three times independently, and each group in each independent repeated experiment was repeated three times, and the results were averaged.

[0062] Example 3. Regulation of AhR signaling pathway activity in human renal tubular epithelial cells (HKC) The steps of HKC cell inoculation, drug treatment, total RNA extraction, and real-time fluorescence quantitative analysis were the same as those in Example 2. 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: AhRR-F: 5'-AGGTTTGGTTGGCAGGACT-3' (SEQ ID NO: 3); AhRR-R: 5'-GCTCAGATGGTTGGCTGTTC-3' (SEQ ID NO: 4); CYP1A1-F: 5'-TCGGCCACGGAGTTTCTC-3' (SEQ ID NO: 5); CYP1A1-R: 5'-GGTCAGCATGTGCCCAATCA-3' (SEQ ID NO: 6); CYP1B1-F: 5'-CTCTGCTGGTCAGGTCCTTG-3' (SEQ ID NO: 7); CYP1B1-R: 5'-CACTGCCAACACCTCTGCT-3' (SEQ ID NO: 8); GAPDH-F: 5'-CATGAGAAGTATGACAACAGCCT-3' (SEQ ID NO: 9); GAPDH-R: 5'-AGTCCTTCCACGATACCAAAGT-3' (SEQ ID NO: 10).

[0063] We evaluated the activity regulation of the AhR signaling pathway by detecting the expression of AhR downstream genes AhRR (NCBI Reference Sequence: NM_001377236, updated: 2024-10-29), CYP1A1 (NCBI Reference Sequence: NM_000499, updated: 2024-10-15), and CYP1B1 (NCBI Reference Sequence: NM_000104, updated: 2024-10-12). Figure 3 As shown in Figure 10, TCDD can increase the expression of AhRR, CYP1A1, and CYP1B1. -8 After 48 hours of MTCDD treatment, the expression levels of AhRR, CYP1A1, and CYP1B1 were 10.26, 43.34, and 5.28 times higher than those of the control group, respectively. The results showed that TCDD can stably and effectively activate the AhR signaling pathway (downstream signaling pathway) in HKC. This experiment was repeated three times independently, and each group in each independent repeated experiment had three replicates.

[0064] Example 4. Effect of regulating the activity of AhR signaling pathway on human renal tubular epithelial-mesenchymal transition HKC cells were cultured at 2×10 5 Cells were inoculated into 6-well plates at a concentration of 100 / mL, with 2 mL of cell suspension per well. Incubate overnight and wait for cells to adhere to the wall. Remove the 6-well plate, aspirate the original culture medium, and wash once with PBS. The experiment was divided into the following groups: Control group: 2 mL / well of low serum medium containing 0.01% DMSO was added to the 6-well plate containing adherent cells for pretreatment. After the cells were pretreated for 3 hours, the original culture medium was aspirated and 2 mL / well of low serum medium containing 0.1% DMSO was added again; 10 -8 M TCDD treatment group: 2 mL / well of low serum medium containing 0.01% DMSO was added to the 6-well plate containing adherent cells for pretreatment. After the cells were pretreated for 3 hours, the original culture medium was aspirated and 10 -8 M TCDD low serum medium 2 mL / well; CH223191 treatment group: 6-well plates containing adherent cells were added with 10 -5 Pre-treat with 2 mL / well of low serum medium of M CH223191. After pre-treating the cells for 3 hours, aspirate the original culture medium and add 2 mL / well of low serum medium containing 0.1% DMSO; 10 -8M TCDD+CH223191 treatment group: 10 -5 Pre-treat the cells with 2 mL / well of low serum medium containing M CH223191. After pre-treating the cells for 3 hours, remove the original culture medium and add 10 -8 2 mL / well of low-serum medium containing M TCDD.

[0065] After culturing the cells for 48 hours, total RNA was extracted for real-time fluorescence quantitative analysis. -8 MTCDD-treated group and 10 -8 The expression of fibrosis marker genes Fibronectin (NCBI Reference Sequence: NM_001365522.2, updated: 2024-11-12), MMP9 (NCBI Reference Sequence: NM_004994.3, updated: 2024-10-29) and IL-6 (NCBI Reference Sequence: NM_001318095.2, updated: 2024-11-12) in the M TCDD+CH223191 treated group was used to evaluate the fibrosis of HKC cells. The extraction of total RNA, real-time fluorescence quantitative analysis and other steps were the same as in Example 2. 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: Fibronectin-F: 5'-GGCTTGAACCAACCTACGGATGAC-3' (SEQ ID NO: 11); Fibronectin-R;5'-TCCTTCTGCCACTGTTCTCCTACG-3' (SEQ ID NO: 12); MMP9-F;5'-AGACCTGGGCAGATTCCAAAC-3' (SEQ ID NO: 13); MMP9-R: 5'-CGGCAAGTCTTCCGAGTAGT-3' (SEQ ID NO: 14); IL-6-F: 5'-TCCTTCTCCACAAACATGTAACAA-3' (SEQ ID NO: 15); IL-6-R: 5'-CCATCTTTGGAAGGTTCAGGTTG-3' (SEQ ID NO: 16); GAPDH-F: 5'-CATGAGAAGTATGACAACAGCCT-3' (SEQ ID NO: 17); GAPDH-R: 5'-AGTCCTTCCACGATACCAAAGT-3' (SEQ ID NO: 18); The fibrosis of HKC cells was evaluated by detecting the expression of fibrosis marker genes Fibronectin, MMP9 and IL-6. Figure 4 As shown in Figure 10, TCDD can increase the expression of Fibronectin, MMP9 and IL-6. -8 After TCDD treatment of cells for 48 hours, the expression levels of Fibronectin, MMP9 and IL-6 were 1.93 times, 2.39 times and 10.01 times higher than those in the control group, respectively. The above results indicate that TCDD can stably increase the expression of specific epithelial-mesenchymal transition marker genes Fibronectin, MMP9 and IL-6 in HKC cells.

[0066] like Figure 5 As shown in the figure, after adding the AhR pathway antagonist CH223191, the expression of epithelial-mesenchymal transition marker genes induced by TCDD in HKC cells was reversed, indicating that CH223191 blocked the epithelial-mesenchymal transition process of HKC cells induced by TCDD by regulating the AhR signaling pathway. This experiment was repeated three times independently, and each group in each independent repeated experiment had three replicates.

[0067] 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 upregulating, improving or increasing the expression of fibrosis marker genes in renal tubular epithelial cells, characterized in that: The method comprises contacting isolated renal tubular epithelial cells with an agonist of aryl hydrocarbon receptor to upregulate, improve or increase the expression of fibrosis marker genes in the renal tubular epithelial cells.

2. A method for preparing target renal tubular epithelial cells with upregulated, enhanced or increased expression of fibrosis marker genes, characterized in that: The method comprises contacting isolated renal tubular epithelial cells with an agonist of aryl hydrocarbon receptor to obtain target renal tubular epithelial cells.

3. The method according to claim 1 or 2, characterized in that: The agonist of the aryl hydrocarbon receptor is dioxin, Furthermore, the dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin.

4. The method according to claim 3, characterized in that The content of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the system in which the renal tubular epithelial cells are contacted with an agonist of an aryl hydrocarbon receptor (AhR) is 10 -10 M, 10 -9 M, 10 -8 M or 10 -7 M, Furthermore, the content of 2,3,7,8-tetrachlorodibenzo-p-dioxin is 10 -8 M.

5. 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.

6. The cell model according to claim 5, characterized in that In the method for preparing the cell model, the agonist of the aromatic hydrocarbon receptor is a dioxin compound, Furthermore, the dioxin compound is 2,3,7,8-tetrachlorodibenzo-p-dioxin.

7. The cell model according to claim 6, characterized in that The content of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the system in which the renal tubular epithelial cells are contacted with an agonist of an aromatic hydrocarbon receptor is 10 -10 M, 10 -9 M, 10 -8 M or 10 -7 M, Furthermore, the content of 2,3,7,8-tetrachlorodibenzo-p-dioxin is 10 -8 M.

8. Use of a substance, characterized in that The substance is a substance that regulates the activity of the AhR signaling pathway, and the use is the use of the substance in preparing a drug for regulating the process of renal tubular epithelial cells to epithelial-mesenchymal transformation.

9. The use according to claim 8, characterized in that The substance is an agonist of the aryl hydrocarbon receptor and / or an antagonist of the aryl hydrocarbon receptor, Furthermore, the aryl hydrocarbon receptor agonist is dioxin, Further, the dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin; Furthermore, the antagonist of aryl hydrocarbon receptor is CH223191.

10. The use according to claim 8 or 9, characterized in that The effective dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin is 10 -10 M, 10 -9 M, 10 -8 M or 10 -7 M, Furthermore, the effective dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin is 10 -8 M; The effective dose of CH223191 is 10 -5 M.

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