Application of TrkB gene in preparation of products for treating hepatic fibrosis

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a TrkB gene in preparation of a hepatic fibrosis treatment product. Through extensive and in-depth research, it is found for the first time that the expression level of hepatocyte TrkB is closely related to the progress of hepatic fibrosis, and in-vitro and in-vivo experiments prove that the over-expression of TrkB can obviously block and reverse the progress of hepatic fibrosis. On the basis, the invention provides a brand new application of the TrkB gene in preparation of a hepatic fibrosis treatment product, and the TrkB gene has important significance in treatment of hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of TrkB gene in preparing a product for treating liver fibrosis. Background Art

[0002] Liver fibrosis is a key intermediate link in the progression and deterioration of many early chronic liver diseases. Further deterioration can lead to serious consequences such as cirrhosis and hepatocellular carcinoma (HCC). Globally, chronic liver disease remains an important health hazard, with more than 2 million people dying from the disease each year. The progression of liver fibrosis mainly includes four stages: ① hepatocyte damage; ② inflammatory cell activation and release of inflammatory factors; ③ activation of hepatic stellate cells (HSCs) and intrahepatic myofibroblasts (MFs); ④ increased secretion of extracellular matrix (ECM) and fibrous scar formation. Blocking the progression of liver fibrosis by targeting different targets in the above stages is a current research hotspot. In recent years, there have been some studies aimed at exploring the therapeutic effects of specific gene therapy on liver fibrosis. For example, it was reported that mixed kinase-like protein (MLKL) is involved in programmed necrosis of liver cells under pathological conditions and is significantly correlated with the degree of liver fibrosis. Gene therapy targeting MLKL in liver parenchymal cells (AAV8-TBG-shMLKL) can effectively alleviate CCl4-induced mouse fibrosis. However, there is still no gene therapy that can reverse liver fibrosis approved for clinical use.

[0003] Therefore, it is of great scientific and social significance to explore specific genes closely related to liver fibrosis and use them as specific regulatory targets to effectively treat liver fibrosis. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a use of the TrkB gene in preparing a product for treating liver fibrosis, so as to solve the problems in the prior art.

[0005] To achieve the above-mentioned object and other related objects, the present invention first provides the use of TrkB gene, TrkB protein or TrkB gene overexpression reagent in preparing liver fibrosis diagnosis products or treatment products.

[0006] The present invention also provides a method for treating liver fibrosis, the method comprising administering a TrkB gene, a TrkB protein or a TrkB overexpression agent to a subject.

[0007] The present invention also provides a composition for preventing or treating liver fibrosis, wherein the effective substance thereof comprises a TrkB overexpression agent and a pharmaceutically acceptable carrier or auxiliary material.

[0008] As described above, the use of the TrkB gene of the present invention in preparing a product for treating liver fibrosis has the following beneficial effects:

[0009] 1) It was first proposed that the expression level of TrkB in hepatocytes is closely related to the progression of liver fibrosis;

[0010] 2) Using multiple in vitro and in vivo models, we first studied and confirmed the blocking and reversing effects of TrkB on the progression of liver fibrosis;

[0011] 3) Hepatocytes account for more than 80% of liver tissue cells. Compared with targeting HSCs, the treatment targeting hepatocytes is more feasible;

[0012] 4) There are more treatment methods for overexpressing TrkB than for downregulating the gene, including recombinant protein, mRNA, gene editing, gene transfer, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shown is a schematic diagram of the difference in expression of TrkB in the livers of patients with different degrees of liver fibrosis in the present invention; Figure 1 A shows H&E, picrosirius red staining, and TrkB immunohistochemistry images of the liver of patients with no significant liver fibrosis (S0-1) and significant liver fibrosis (S2-4); Figure 1 B shows the statistical graph of Sirius red and TrkB immunohistochemical analysis of livers of patients with no significant fibrosis and significant fibrosis (n=4); Figure 1 C shows the correlation analysis diagram between the Sirius red positive area and the TrkB H-score.

[0014] Figure 2 It is a schematic diagram showing the difference in expression of TrkB in the liver of control mice and GAN-fed OBOB mice in the present invention; Figure 2 A shows a schematic diagram of the construction of the liver fibrosis model of GAN-fed OBOB mice; Figure 2 B shows the expression of TrkB in primary hepatocytes isolated from the livers of control group and GAN-fed OBOB mice detected by WB (n=3) and qPCR (n=4).

[0015] Figure 3 Shown is a schematic diagram of the difference in expression of TrkB in control and PA-treated hepatocytes in the present invention; Figure 3 A shows a schematic diagram of the in vitro MASH model simulated by PA-treated hepatocytes; WB and qPCR were used to detect the expression of TrkB in the control group and PA-treated hepatocytes (n=4).

[0016] Figure 4 Shown is a schematic diagram of the difference in expression of TrkB in the physiological group and the MASH-3D model in the present invention; Figure 4 A shows a schematic diagram of MASH 3D model construction; Figure 4 B shows the oil red staining and TrkB immunofluorescence images of 3D spheroids in the physiological group and MASH group.

[0017] Figure 5 Shown is a schematic diagram of the inhibition of liver inflammation and fibrosis by overexpression of TrkB in hepatocytes in 3D organoids of the present invention; Figure 5 A shows a schematic diagram of the construction of a 3D liver fibrosis model; Figure 5 B shows H&E, oil red staining, picrosirius red staining, and αSMA immunohistochemistry of control spheroids and hepatocytes overexpressing TrkB spheroids; Figure 5 C is displayed as Figure 5 Statistical graph of immunohistochemical analysis of oil red, picrosirius red and αSMA in liver ( n = 3) in B; Figure 5 D shows the concentrations of IL-6, TNF-α, CCL2, and TGF-β in the supernatant of control spheres and hepatocytes overexpressing TrkB detected by ELISA (n=5).

[0018] Figure 6 Schematic diagram showing that overexpression of hepatocyte TrkB alleviates the progression of liver fibrosis in mice; Figure 6 A shows a schematic diagram of the liver fibrosis model of AAV8-injected OBOB mice; Figure 6 B shows H&E, oil red staining, picrosirius red staining, and αSMA immunohistochemistry of the liver of AAV8-control and AAV8-TrkB mice; Figure 6 C is displayed as Figure 6 Oil red, picrosirius red and αSMA immunohistochemical analysis of liver in B ( n = 5); Figure 6 D shows the triglyceride content in the liver of AAV8-control and AAV8-TrkB mice (n=5); Figure 6 E shows the mRNA levels of Il6, Il1b, Tnfα, and Ccl2 in the livers of AAV8-control and AAV8-TrkB mice detected by qPCR (n=6); Figure 6 F shows the mRNA levels of Col1a1, Col3a1, Ctgf, Tgfb, and Acta2 in the livers of AAV8-control and AAV8-TrkB mice detected by qPCR (n=6); Figure 6 G shows the concentrations of IL-6, TNF-α, CCL2, and TGF-β in the serum of AAV8-control and AAV8-TrkB mice detected by ELISA (n=6). DETAILED DESCRIPTION

[0019] The present invention proposes for the first time that the expression level of TrkB in hepatocytes is closely related to the progression of liver fibrosis.

[0020] The present invention firstly provides the use of TrkB gene, TrkB protein or TrkB gene overexpression reagent in preparing liver fibrosis diagnosis product or treatment product.

[0021] In the present invention, the product is selected from one or more of medicines, health products or foods.

[0022] In certain embodiments of the present invention, the TrkB gene is a TrkB gene fragment or a TrkB full-length sequence.

[0023] In certain embodiments of the present invention, the TrkB protein is a complete TrkB protein or a protein fragment; the TrkB protein is a natural TrkB protein or a recombinant TrkB protein.

[0024] In certain embodiments of the present invention, the TrkB overexpression agent is selected from a nucleic acid construct of the TrkB gene or a virus with the TrkB gene integrated into its genome.

[0025] In certain embodiments of the present invention, the nucleic acid construct comprising the TrkB gene can be obtained by cloning a TrkB gene fragment into a known vector.

[0026] In certain embodiments of the present invention, the TrkB gene nucleic acid construct is a mammalian expression vector.

[0027] Furthermore, the mammalian expression vector is selected from one or more of a TrkB gene adeno-associated virus vector, a TrkB gene lentivirus vector, a TrkB gene adenovirus vector or a TrkB gene retrovirus vector.

[0028] Furthermore, the adeno-associated virus vector is AAV8.

[0029] Furthermore, the lentiviral vector is pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PUR.

[0030] In the embodiments of the present invention, an AAV8 vector containing a TrkB gene and a lentiviral vector containing a TrkB gene are specifically constructed. The TrkB gene is shown in SEQ ID NO.1. The TrkB gene lentiviral vector or TrkB gene adeno-associated virus AAV8 vector disclosed in the present invention is obtained by cloning a TrkB gene fragment into a known vector, wherein the known vector is a lentiviral vector or an adeno-associated virus AAV8 vector, and the TrkB gene lentiviral vector or TrkB gene adeno-associated virus AAV8 vector is packaged into infectious virus particles by the virus, and then infects liver cells, thereby overexpressing the TrkB gene.

[0031] In certain embodiments of the present invention, the virus into which the TrkB gene is integrated is selected from one or more of lentivirus, adenovirus, adeno-associated virus or retrovirus.

[0032] The expression level of TrkB gene in liver tissue was detected by immunohistochemistry. The study found that the expression level of TrkB in liver tissue was significantly lower than that in normal tissue. This suggests that the expression level of TrkB gene may become a marker for the diagnosis of liver fibrosis.

[0033] The liver fibrosis therapeutic drug is a molecule that can specifically promote the transcription or translation of the TrkB gene, or can specifically promote the expression of the TrkB gene or the activity of the TrkB protein, thereby increasing the content level of the TrkB protein in the patient's body.

[0034] The liver fibrosis treatment drugs or liver fibrosis diagnosis drugs prepared by TrkB gene, TrkB protein or TrkB gene overexpression reagent include but are not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins, adeno-associated viruses or lentiviruses.

[0035] The dosage of the liver fibrosis treatment drug is sufficient to upregulate the transcription or translation of the TrkB gene, or the expression or activity of the TrkB protein, so that the expression of the TrkB gene is at least upregulated by 50%, 80%, 90%, 95% or 99%.

[0036] TrkB overexpression reagent refers to a substance that has an upregulating effect on TrkB. The upregulating effect on TrkB includes but is not limited to:

[0037] 1) Promote the expression of TrkB gene or the activity of TrkB protein;

[0038] 2) using overexpression to increase the expression level of the TrkB gene;

[0039] 3) Directly increase the level of TrkB gene in liver cells.

[0040] Promoting the activity of TrkB protein means increasing the activity of TrkB protein. Preferably, compared with before the increase, the activity of TrkB protein increases by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0041] Promoting TrkB gene expression may specifically be promoting the transcription or translation of TrkB gene, and specifically, may mean: increasing the gene transcription or expression of TrkB by at least 10%, preferably by at least 30%, more preferably by at least 50%, more preferably by 70%, and most preferably by at least 90%.

[0042] Use of TrkB overexpression reagent in the preparation of products having any of the following effects:

[0043] 1) Treatment of liver fibrosis;

[0044] 2) inhibiting the mRNA expression of inflammation- or fibrosis-related genes; specifically, the inflammation- and fibrosis-related genes include one or more of Il6, Il1b, Tnf, Tgfb, Ccl2, Col3a1, Col1a1, Acta2 or CTGF;

[0045] 3) reducing the concentration of inflammatory factors or fibrosis factors; specifically, the inflammatory factors are IL-6, TNF-α, and CCL2;

[0046] The fibrosis factor is TGF-β.

[0047] The up-regulation of TrkB gene expression can be verified by detecting the expression level by PCR and Western Blot.

[0048] In certain embodiments of the present invention, the expression level of TrkB can be increased by transfecting an overexpression plasmid.

[0049] In certain embodiments of the present invention, the expression level of TrkB can be increased by constructing a lentiviral vector.

[0050] In certain embodiments of the present invention, the expression level of TrkB can be increased by constructing an adeno-associated virus vector.

[0051] The liver fibrosis treatment drug must include TrkB gene, TrkB protein or TrkB overexpression agent, and uses TrkB gene, TrkB protein or TrkB overexpression agent as the effective ingredient for the above functions.

[0052] In the liver fibrosis treatment drug, the effective ingredient that exerts the aforementioned function may be only the TrkB gene, TrkB protein or TrkB overexpression agent, or may contain other molecules that can exert similar functions.

[0053] The TrkB gene, TrkB protein or TrkB overexpression agent is the only effective ingredient or one of the effective ingredients of the liver fibrosis treatment drug.

[0054] The liver fibrosis treatment drug may be a single-component substance or a multi-component substance.

[0055] The form of the liver fibrosis therapeutic drug is not particularly limited, and can be in various forms such as solid, liquid, gel, semi-fluid, aerosol, etc.

[0056] In certain embodiments of the present invention, the product is a liver fibrosis treatment product that acts on liver cells.

[0057] The product is mainly targeted at mammals. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes or humans.

[0058] The products include but are not limited to medicines, health products, foods, etc.

[0059] In certain embodiments of the present invention, the diagnostic product is a diagnostic kit.

[0060] In certain embodiments of the present invention, the diagnostic kit is used to quantitatively detect the level of TrkB protein.

[0061] In certain embodiments of the present invention, the test sample of the diagnostic kit is liver tissue, hepatocytes or hepatic stellate cells.

[0062] The present invention also provides a method for treating liver fibrosis, the method comprising administering a TrkB gene, a TrkB protein or a TrkB overexpression agent to a subject.

[0063] The subject may be a mammal. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. The primate is preferably a monkey, an ape or a human.

[0064] The subject may be a patient suffering from liver fibrosis or an individual who expects to prevent or alleviate liver fibrosis. Alternatively, the subject may be an ex vivo liver tissue, hepatocyte or hepatic stellate cell of a patient suffering from liver fibrosis or an individual who expects to treat liver fibrosis.

[0065] The TrkB gene, TrkB protein or TrkB overexpressing agent can be administered to a subject before, during or after receiving treatment for liver fibrosis.

[0066] The use of the drug for preventing or treating liver fibrosis provides a method for treating liver fibrosis, specifically a method for preventing or treating liver fibrosis in a subject, comprising administering an effective dose of the drug to the subject.

[0067] The subject of the method may be a human.

[0068] The present invention also provides a composition for preventing, diagnosing or treating liver fibrosis, wherein the effective substance contains TrkB gene, TrkB protein or TrkB overexpression agent and pharmaceutically acceptable carrier or auxiliary material.

[0069] The drug for preventing, diagnosing or treating liver fibrosis must include TrkB gene, TrkB protein or TrkB overexpression agent, and use TrkB gene, TrkB protein or TrkB overexpression agent as the active ingredient for the aforementioned function.

[0070] "Pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to an animal or a human.

[0071] "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0072] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0073] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0074] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0075] In the present invention, all data are expressed as mean ± standard error (SEM). Statistical analysis was performed using SPSS20.0 software. When comparing two groups of data, a normal distribution test was performed first, and then a student's t test was used. If normal distribution or homogeneity of variance was not satisfied, the Mann-Whitney U test was used to compare the two groups. When the experimental design involved comparisons of more than two groups, a normal distribution test was performed first, and then analysis of variance (ANOVA) was used to compare differences between the groups, and the least significant difference (LSD) test was used for multiple comparisons between the two groups. If normal distribution or homogeneity of variance was not satisfied, the Kruskal-Wallis test was used to compare differences between the groups, and the Hodges-Lehmann test was used for multiple comparisons between the two groups. A p value of less than 0.05 was considered to be statistically significant. P values ​​are represented by *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0076] Example 1

[0077] To clarify the relationship between hepatic TrkB expression and the progression of liver fibrosis, clinical liver biopsy samples from patients with metabolic dysfunction-associated steatohepatitis (MASH)-related liver fibrosis were collected. The paraffin sections were from patients with chronic liver disease who underwent liver puncture in Zhongshan Hospital of Fudan University, which was approved by the Ethics Committee of Zhongshan Hospital of Fudan University, and all patients signed informed consent.

[0078] 1. The steps for H&E staining of sections are as follows:

[0079] 1) Dewax the sections by soaking them in xylene for 5 minutes twice;

[0080] 2) Rehydrate in a gradient ethanol solution, passing through 100%, 95%, 90%, and 70% ethanol solutions in sequence, staying at each level for about 5 minutes, and finally soaking in distilled water for 5 minutes;

[0081] 3) Stain the sections in hematoxylin stain for 5-15 minutes, then rinse with running water for 15-30 minutes to make the nuclei blue, and differentiate the sections in 1% hydrochloric acid alcohol solution for 1-3 seconds to remove excess hematoxylin stain;

[0082] 4) Stain the sections in 0.5% eosin solution for 1-5 minutes, then quickly wash off the excess eosin solution with 95% and 100% ethanol solutions;

[0083] 5) Then transparentize the sections in xylene, immerse for 5 minutes each time, twice;

[0084] 6) Use neutral gum to seal the slides.

[0085] 2. Stain the sections with Sirius red as follows:

[0086] 1) Add iron hematoxylin staining solution to the sections for 5-10 minutes, then wash with distilled water for 10-20 seconds to remove excess staining solution;

[0087] 2) Then wash the slices with tap water for 5 minutes;

[0088] 3) Add Sirius red staining solution to the slices for 15-30 minutes, and rinse the slice surface with light running water;

[0089] 4) Dehydrate the sections in 75% ethanol for 1 minute, then in 95% ethanol for 1 minute, then in anhydrous ethanol for 1 minute, and then in xylene for 3 times, 1-2 minutes each time;

[0090] 5) Use neutral gum to seal the sections.

[0091] 3. Use immunohistochemistry to stain liver sections for TrkB. The steps are as follows:

[0092] 1) Dewax the paraffin-embedded tissue sections to water, dewax the sections in xylene for 10 minutes, then pass through 100%, 95%, and 70% ethanol solutions for 5 minutes each, and finally rinse with distilled water for 5 minutes;

[0093] 2) Incubate the sections in 3% hydrogen peroxide solution for 10 minutes to block endogenous peroxidase activity; wash the sections three times with PBS buffer, 3 minutes each time;

[0094] 3) Place the slices in a solution containing TRKB protein-specific primary antibody (Santa Cruz, 1:50) and incubate overnight at 4°C;

[0095] 4) The next day, wash the sections three times with PBS buffer, 3 minutes each time; add biotin-labeled secondary antibody corresponding to the primary antibody and incubate at room temperature for 30 minutes; wash the sections three times with PBS buffer again, 3 minutes each time;

[0096] 5) Add streptavidin-peroxidase conjugate and incubate at room temperature for 20 minutes; wash the sections 3 times with PBS buffer, 3 minutes each time;

[0097] 6) After adding DAB color developer for 5-10 minutes, rinse the sections with tap water to terminate the color development reaction;

[0098] 7) Place the sections in hematoxylin for 1 minute, differentiate with 1% hydrochloric acid alcohol for 10 seconds, and then rinse with tap water to turn blue;

[0099] 8) Finally, the sections were dehydrated in gradient ethanol solutions, transparentized with xylene, and sealed with neutral gum.

[0100] 11. Microscope observation and data analysis: Use bright field microscopy to observe H&E and Sirius Red stained sections; use fluorescence microscopy or enzyme-labeled microscopy to observe TrkB immunohistochemically stained sections; use Image-J software to perform photometric semi-quantitative analysis of Sirius Red and TrkB positive areas. Figure 1 As shown in A.

[0101] 12. Results: Through the above experiments, we found that as the degree of liver fibrosis increases, the expression of TrkB in liver hepatocytes decreases, and there is a negative correlation between the expression level of TrkB and the severity of fibrosis ( Figure 1 AC).

[0102] Example 2

[0103] We further established a GAN-fed OB / OB mouse MASH model with the following steps: 8-week-old male OB / OB mice were fed a diet containing Gubra-Amylin MASH (GAN) (D09100310, Research Diets, NJ, USA) for 16 weeks.

[0104] The method for extracting primary mouse hepatocytes is as follows: primary mouse cells are isolated using collagenase perfusion. After perfusion, the liver is removed by cutting off the blood vessels and mesangial structures, the liver capsule structure is torn open, and the liver cells are separated and filtered using 1640 culture medium in a 70-micron filter membrane. After centrifugation at 4°C and 50g for 2 minutes, the precipitate contains hepatocytes and the supernatant contains non-parenchymal cells, which are collected separately. The cells are stained with trypan blue and counted by a blood cell counting chamber.

[0105] RTq-PCR was used to detect the mRNA expression level of TrkB in the liver hepatocytes of the two groups of mice as follows:

[0106] 1) Centrifuge at 300g for 5 minutes at 4°C, discard the supernatant of mouse primary hepatocytes, and wash once with 1× PBS. 6 Add 500 μl RNA easy solution to each cell. Use a pipette to repeatedly pipette until cells are fully lysed.

[0107] 2) Add 2 / 5 volume of RNase-free ddH2O to the above lysate (200 μl for every 500 μl RNA-easy), mix by inversion, and let stand at room temperature for 5 min;

[0108] 3) Centrifuge at 11,200 rpm (12,000 × g) for 15 min at room temperature;

[0109] 4) Take out the centrifuge tube and transfer the upper aqueous phase to a new centrifuge tube. Add an equal volume of isopropanol, mix by inverting, and let stand at room temperature for 10 minutes;

[0110] 5) Centrifuge at 11,200 rpm (12,000 × g) for 10 min at room temperature and discard the supernatant;

[0111] 6) Add 500 μl 75% ethanol (RNase-free ddH2O), suspend the precipitate, and invert several times. 9,100

[0112] rpm (8,000 × g) at room temperature for 3 min, and the supernatant was discarded;

[0113] 7) After the precipitate is dried, it is fully dissolved in an appropriate amount of RNase-free ddH2O and quantified using an ultra-micro spectrophotometer. The A260 / A280 ratio between 1.8 and 2.0 indicates that the RNA purity is qualified. It is then stored in a -80°C refrigerator for later use;

[0114] 8) The ABScript II cDNA First-Strand Synthesis Kit was used, and the specific composition is shown in Table 1:

[0115] Table 1 Reverse transcription system composition

[0116]

[0117] After the configuration is completed, place it in the gradient PCR instrument, and the reverse transcription program settings are shown in Table 2:

[0118] Table 2 Reverse transcription program settings

[0119]

[0120] After reverse transcription, the cDNA samples were stored in a -80°C refrigerator for later use;

[0121] 9) Using Yeason Hieff Universal Blue qPCR SYBR Master Mix Kit, the specific composition is shown in Table 3:

[0122] Table 3 qRT-PCR system composition

[0123]

[0124]

[0125] Three technical replicates were set for each sample, and the system was prepared in a 96-well PCR microplate and tested in a fluorescent quantitative PCR instrument. The reaction procedure is shown in Table 4:

[0126] Table 4 qRT-PCR program settings

[0127]

[0128] GAPDH was used as the internal reference in the samples. -ΔΔCt The expression level of mRNA was evaluated by Δ Ct=Ct(target mRNA)-Ct(internal reference), ΔΔ Ct= Δ Ctn- Δ Ct1 (n is the sample number).

[0129] The primers of the present invention were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. The qRT-PCR primer sequence information is shown in Table 5:

[0130] Table 5 qRT-PCR primer sequence information

[0131]

[0132] The expression level of TrkB protein in the hepatocytes of the liver of the two groups of mice was detected by western blot experimental technology. The method was as follows: The total protein of the hepatocytes was isolated and extracted from the mouse liver samples, and RIPA lysis buffer containing protease inhibitors was used. The protein concentration was measured using a BCA protein quantification kit. A 10% polyacrylamide gel was made to separate each protein, and the separated proteins were transferred to a PVDF membrane. After blocking with a protein-free rapid blocking buffer at room temperature for 15 minutes, the membrane was incubated with a TrkB primary antibody (Abcam, 1:1000) at 4°C overnight, followed by incubation with the corresponding secondary antibody at room temperature for 60 minutes. Blot imaging was performed using the ChemiDoc MP imaging system (Bio-Rad). All results were performed in three independent experiments.

[0133] like Figure 2 As shown, by comparing the hepatocytes of the liver of MASH model mice and normal control mice, it was found that TrkB expression in the hepatocytes of MASH mice was significantly downregulated.

[0134] Example 3

[0135] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 atmosphere. HepG2 cells were treated with palmitic acid (PA), which is known to induce fatty degeneration and fibrosis, at a concentration of 0.5 mM for 48 hours to simulate fatty degeneration and inflammatory lesions of hepatocytes at the 2D level in vitro. The expression level of TrkB in the two groups of cells was detected by RTq-PCR and western blot experimental techniques, using the same method as above. Figure 3 As shown, it was found that HepG2 cells showed decreased TrkB expression at both mRNA and protein levels after PA treatment.

[0136] Example 4

[0137] In the present invention, a 3D liver organoid MASH model was constructed, and the construction of the organoid model was based on patent CN118388568A. First, a DNA origami platform (DOP) cell linker consisting of a long-chain DNA scaffold M13mp18 and 242 single-stranded DNAs was designed based on the hybridization characteristics of DNA nanomaterials. Then, human primary hepatocytes were assembled with paired DOP cell linkers to generate spherical nuclei, and exposed to 0.5mM palmitic acid-oleic acid (PA-OA) for 96 hours to induce fatty and inflammatory changes. Figure 4 As shown, it was found that the 3D hepatic steatosis model containing only hepatocytes also showed reduced expression of TrkB compared with the control group.

[0138] Example 5

[0139] Firstly, a 3D model of MASH-related liver fibrosis composed of hepatocytes and hepatic stellate cells was constructed. Figure 5 A).

[0140] The steps are as follows: First, TrkB is overexpressed in human primary hepatocytes using a lentiviral vector, wherein the lentiviral vector contains a TrkB gene, and the nucleotide sequence of the TrkB gene is shown in SEQ ID NO.1. Then, the human primary hepatocytes transfected with the lentiviral vector are mixed and connected with human primary stellate cells through a DOP cell connector, and a dense spheroid structure is formed after 24 hours. It is then exposed to 0.5mM palmitic acid oleic acid (PA-OA) for 7 days to induce fat and inflammatory changes. All 3D liver organoids contain a total of 3,000 cells per organoid, with a ratio of hepatocytes to stellate cells of 10:1 to simulate the physiological cell composition of the liver.

[0141] Next, the 3D liver organoid spheroids were stained with H&E, picrosirius red, oil red, and αSMA immunofluorescence staining. The steps for H&E and picrosirius red staining were the same as before. The specific steps for oil red staining are as follows:

[0142] 1) First, prepare frozen sections of 3D microspheres. Collect the 3D microspheres into a 1.5 mL EP tube using a 1 mL pipette with a trimmed tip, and fix them with 4% paraformaldehyde for 2 hours. Discard the paraformaldehyde, rinse with PBS 2-3 times, and add 30%

[0143] After the sucrose solution is dehydrated and the pellets gradually sink to the bottom, the sucrose solution is discarded and the pellets are rinsed 2-3 times with PBS.

[0144] Add a small amount of OCT to the bottom of a 10mm × 10mm frozen section mold, place the mass horizontally, and then fill it with OCT. Place the mold in liquid nitrogen for rapid freezing and store it at -80℃ for long-term storage;

[0145] 2) When slicing, remove the mold and place it on the freezing table of the cryostat for 30 minutes. Fix the embedded block on the slicing table, set the slice thickness to 8μm, and adjust the angle between the slicer head and the slicing table to the appropriate position. Use a brush to carefully paste the cut frozen section onto a clean slide and store it at 4℃ for long-term storage;

[0146] 3) Take the frozen sections out of the 4°C refrigerator and equilibrate at room temperature for 30 minutes. Preheat PBS at 37°C and rinse the sections until the OCT is removed. Use absorbent paper to absorb excess water and circle the area to be stained with a hydrophobic pen. Add 0.5g of oil red powder to 100mL of isopropanol to prepare the oil red staining stock solution. When using, dilute it with sterile water at a ratio of 3:2. Stain the oil red staining solution at room temperature for about 5 minutes and observe under the microscope at any time. After washing off the dye, it can be briefly differentiated with 75% ethanol.

[0147] After washing, co-stain with hematoxylin for 5 minutes; seal the slices with sealing medium and store them at room temperature.

[0148] The steps of αSMA immunofluorescence staining are as follows:

[0149] 1) After the frozen sections are taken out of the 4°C refrigerator, they are equilibrated at room temperature for 30 minutes. Preheat PBS at 37°C and rinse the sections until the OCT is removed. Use absorbent paper to absorb excess water and circle the area to be stained with a hydrophobic pen.

[0150] 2) Cover the stained area with 10% goat serum and block at room temperature for 30 minutes;

[0151] 3) Add 0.5 mL of Tween20 to 500 mL of 1×PBS to prepare a washing solution. Rinse the sections thoroughly with the washing solution for 5 minutes, then soak in PBS for 3 minutes and absorb excess water with absorbent paper.

[0152] 4) Incubate with 1:100 diluted αSMA primary antibody (Abclonal, 1:50) in the dark for 1 hour, wash away the primary antibody with washing solution for at least 5 minutes three times, and then rinse with PBS for 3 minutes;

[0153] 5) Incubate with 1:100 diluted secondary antibody in dark for 1 hour, wash off the secondary antibody with water for at least 5 minutes three times, and then rinse with PBS for 3 minutes;

[0154] 6) Dilute DAPI stain at 1:100, stain the cell nucleus for 5 minutes, then wash thoroughly with PBS and remove excess water;

[0155] 7) Seal the slides with anti-fade mounting medium and store them at 4°C.

[0156] Image-J software was used to perform semi-quantitative photometric analysis of the positive areas of picrosirius red and αSMA.

[0157] like Figure 5 As shown in BC, in organoids with overexpression of TrkB in hepatocytes, the degree of steatosis and fibrosis was significantly reduced.

[0158] Subsequently, the ELISA experiment was used to detect the content of four cytokines, IL-6, TNF-α, CCL2 and TGF-β, in the supernatant of 3D liver organoid spheroids. The specific steps are as follows: Dilute the supernatant sample 1:2 or 1:4. Use 1mL PT 1-ef sample diluent to reconstitute the standard. Set up zero well, standard well, and sample well to be tested respectively. Add 100μL of sample diluent to zero well, and add 100μL / well of graded diluted standard or sample to be tested to the remaining wells. Cover the standard plate with a film and incubate at 37℃ for 2h. Wash the strips with washing solution, 350-400μL per well. After washing, pat the plate dry and repeat this step 4 times. Add 100μL of detection antibody (1×) to each well, cover with a sealing film, incubate at 37℃ for 1h, and then wash the plate. Add 100μL of HRP-labeled streptavidin to each well, cover with a sealing film, incubate at 37℃ for 40min, and then wash the plate. Add 100 μL of TMB colorimetric solution to each well and color at 37°C in the dark for 15-20 min. Add 100 μL of stop solution to each well. Use 630 nm as the calibration wavelength and use an ELISA reader to measure the optical density of each well at a wavelength of 450 nm.

[0159] like Figure 5 As shown in D, it was found that the secretion of inflammatory and fibrotic factors (IL-6, TNF-α, CCL2 and TGF-β) by liver organoid spheroids overexpressing TrkB was reduced.

[0160] Example 6

[0161] To further verify the therapeutic effect of overexpression of TrkB in hepatocytes on liver fibrosis in vivo, a GAN diet-induced liver fibrosis model was established in OBOB mice, and AAV8 was used to target mouse hepatocytes for TrkB overexpression ( Figure 6 A).

[0162] The construction steps are as follows: 1) Use restriction endonuclease digestion to obtain a linearized AAV8 vector, and PCR amplify to prepare a TrkB gene fragment. The nucleotide sequence of the TrkB gene fragment is shown in SEQ ID NO.1. The amplification primer used needs to add a homologous recombination sequence at its 5' end. The gene fragment is amplified using this primer, and the sequences at the 5' and 3' ends of the amplified product are completely consistent with the sequences at both ends of the linearized vector. Prepare a reaction system with the linearized vector and the gene amplification product, perform a recombination reaction, realize in vitro circularization, and construct an AAV8 vector containing the TRKB gene.

[0163] 2) The constructed AAV8 vector plasmid is co-transfected with the helper plasmid and the packaging plasmid into HEK293 cells to produce recombinant AAV8 virus.

[0164] 3) 72 hours after transfection, the cell supernatant and cell pellet were collected, and the cells were lysed by repeated freezing and thawing to collect the crude virus extract.

[0165] 4) Concentrate and purify the virus using the PEG8000 concentration method or sucrose density gradient ultracentrifugation method.

[0166] 5) Ensure the quality of the virus by detecting virus titer by qPCR and virus purity by Coomassie Brilliant Blue staining.

[0167] 6) Male OB / OB mice aged 8 weeks were fed with GAN diet for 16 weeks. AAV8 vector was injected into the tail vein at the 8th and 12th weeks of feeding at a dose of 1×10 11 vector genomes / gram body weight (VG / g).

[0168] At the end of the modeling, the mouse livers were stained with H&E, oil red, picrosirius red, and αSMA using the same staining methods as before. The triglyceride content in the livers of the two groups of mice was detected using a triglyceride kit as follows:

[0169] 1) After the mouse liver sample was ground and homogenized, it was centrifuged at 2000 rpm for 5 min at room temperature, and the supernatant was taken as the sample for TG detection.

[0170] 2) Mix the test solution of the test kit in the ratio of R1:R2=4:1 and prepare it before use.

[0171] 3) Dilute the standard to 4 mM and prepare standards of different concentrations: 1000 μM, 250 μM, 62.5 μM, 15.625 μM, and 0 μM.

[0172] 4) Add 10 μL of sample, standard, ddH2O (blank), and 190 μL of working solution (sample in 3 replicate wells, standard in 2 replicate wells).

[0173] 5) Incubate at 37°C for 15 min and measure OD at 550 nm.

[0174] 6) Use OD value as the y-axis and standard concentration as the x-axis to obtain the formula and calculate the sample concentration.

[0175] 7) Tissue protein was quantified using a BCA kit and the data was presented as μmol triglyceride per mg protein.

[0176] RTq-PCR technology was used to detect the mRNA expression levels of inflammation and fibrosis-related genes in the livers of the two groups of mice, and ELISA experiments were used to detect the levels of IL-6, TNF-α, CCL2 and TGF-β cytokines in the livers of the two groups of mice, using the same method as before.

[0177] Table 6 qRT-PCR primer sequence information

[0178]

[0179]

[0180] The results showed that overexpression of hepatocyte TrkB could significantly reduce liver steatosis and fibrosis in mice and reduce the triglyceride content in the liver ( Figure 6 BD). It was found that overexpression of TrkB in hepatocytes significantly inhibited the mRNA expression of inflammation and fibrosis-related genes in mouse liver ( Figure 6 EF). ELISA experiments using mouse serum showed that the secretion concentrations of inflammatory factors such as IL-6, TNF-α, CCL2, and fibrosis factor TGF-β in the TrkB overexpression group were significantly reduced ( Figure 6 G).

[0181] Sequences involved in this application:

[0182] SEQ ID NO.1

[0183]

[0184]

[0185] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of TrkB gene, TrkB protein or TrkB gene overexpression reagent in the preparation of a product for the treatment of liver fibrosis.

2. The use according to claim 1, characterized in that Use of the TrkB overexpression reagent in preparing a product having any of the following effects: 1) Treatment of liver fibrosis; 2) inhibiting the mRNA expression of inflammation- or fibrosis-related genes; 3) Reduce the concentration of inflammatory factors or fibrosis factors.

3. The use according to claim 1, characterized in that The TrkB gene is a TrkB gene fragment or a TrkB full-length sequence; and / or, the TrkB protein is a TrkB complete protein or a protein fragment; preferably, the TrkB protein is a natural protein or a recombinant protein.

4. The use according to claim 1, characterized in that The TrkB overexpression agent is selected from a nucleic acid construct comprising a TrkB gene or a virus having a TrkB gene integrated into its genome.

5. The use according to claim 4, characterized in that The nucleotide sequence of the TrkB gene in the nucleic acid construct comprising the TrkB gene or the virus with the TrkB gene integrated into its genome is shown in SEQ ID NO.

1.

6. The use according to claim 4, characterized in that The TrkB gene nucleic acid construct is a mammalian expression vector; preferably, the mammalian expression vector is selected from one or more of a TrkB gene adeno-associated virus vector, a TrkB gene lentivirus vector, a TrkB gene adenovirus vector or a TrkB gene retrovirus vector.

7. The use according to claim 4, characterized in that The virus with the TrkB gene integrated into the genome is any one or more of a TrkB gene lentivirus, a TrkB gene adeno-associated virus, a TrkB gene adenovirus or a TrkB gene retrovirus.

8. The use according to claim 1, wherein the product is a product for treating liver fibrosis that acts on liver cells.

9. The use according to claim 1, wherein the product is selected from one or more of medicines, health products or foods.

10. The use according to claim 1, when the product is a medicine or a health product, the product further comprises a pharmaceutically acceptable carrier or excipient.

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