Application of TINAGL1 in the preparation of anti-hepatic fibrosis drugs and new uses of compounds

Abstract

The present invention belongs to the field of medicine and discloses the use of tubulointerstitial nephritis antigen-like protein 1 (TINAGL1) as a direct target for screening anti-hepatic fibrosis drugs. By using TINAGL1 as a direct target, target drugs that can bind to human TINAGL1 recombinant protein can be screened out. These target drugs have been proven to be able to reduce the increase in the level of the fibrosis factor COL1A1 in LX-2 cells induced by PDGF-BB and can achieve anti-hepatic fibrosis effects to varying degrees. At the same time, the present invention also discloses a screening method for anti-hepatic fibrosis drugs, new potential anti-hepatic fibrosis drugs, and new uses of compounds.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of TINAGL1 in the preparation of anti-hepatic fibrosis drugs and new uses of compounds. Background Art

[0002] Currently, about 800 million people worldwide are troubled by chronic liver diseases, with more than 2 million deaths annually. Among them, advanced liver diseases caused by hepatic fibrosis are an important cause of death, and its incidence is increasing year by year. Hepatic fibrosis is a fibrotic scar formed due to the excessive accumulation of extracellular matrix proteins, mainly manifested as the replacement of damaged normal tissues by type I and type III collagens. At present, some Chinese patent medicines, such as Fuzheng Huayu Capsule, Compound Turtle Soft Liver Tablets, and Anluo Huaxian Pills, can play a certain role in anti-hepatic fibrosis, and drugs targeting some targets, such as hepatic stearoyl-CoA desaturase, chemokine receptor 2 / 5, farnesoid X receptor, nicotinamide adenine dinucleotide phosphate oxidase 3, cyclophilin, fibroblast growth factor, galectin-3, glucagon-like peptide-1, and peroxisome proliferator-activated receptor, etc., are being evaluated for the treatment of hepatic fibrosis, but no specific drugs have been approved for clinical use. Therefore, finding new therapeutic targets for hepatic fibrosis and their therapeutic drugs has important clinical value.

[0003] Platelet-derived growth factor (PDGF) is one of the important mediators of fibrotic diseases and is involved in the regulation of fibrosis in various organs such as the liver, kidney, and heart. PDGF has four subunits, namely PDGF-A, PDGF-B, PDGF-C, and PDGF-D, which can be linked by disulfide bonds to form five polymers with different functions (PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD). Among them, PDGF-AB and PDGF-BB can promote collagen synthesis, and PDGF-BB is the factor most closely related to the activation of early human hepatic stellate cells. After PDGF binds to its receptor PDGFR, it induces receptor dimerization and phosphorylation, which in turn phosphorylates tyrosine residues on different intracellular substrates, thereby triggering the activation of multiple signaling pathways such as the Ras / Raf pathway, the phosphoinositide 3-kinase / protein kinase B pathway, and the Janus kinase / signal transducer and activator of transcription pathway. These downstream elements can further regulate the expression of profibrotic genes such as collagen type I alpha 1 chain (COL1A1), tissue inhibitor of matrix metalloproteinase (TIMP), and matrix metalloproteinases (MMPs), as well as apoptosis regulator B-cell lymphoma-2 gene (Bcl-2), thereby regulating cell proliferation, survival, and the progression of organ fibrosis. The research results show that the expressions of PDGF-BB and its receptor PDGFR are significantly upregulated in animal models of liver fibrosis and the progression of human liver fibrosis diseases. Therefore, regulating the PDGF signal, especially the liver fibrosis activation signal induced by PDGF-BB, may provide new possibilities for the treatment of liver fibrosis.

[0004] TINAGL1 is a newly discovered extracellular matrix protein that plays an important role in the occurrence and development of many tumors. TINAGL1 can bind to fibronectin or collagen and be fixed on the matrix to promote the adhesion and spreading of adrenal cortical cells. Moreover, TINAGL1 can further regulate the adhesion between adrenal cortex and vascular smooth muscle cells through α1β1, α2β3, and α5β4 integrin receptors. Meanwhile, TINAGL1 protein can also directly bind to integrin α5β1, αvβ1, and EGFR, inhibit the focal adhesion kinase and epidermal growth factor receptor signaling pathways, and thus hinder the progression and metastasis of triple-negative breast cancer. In addition, TINAGL1 is significantly upregulated in human liver cancer tissues, can significantly activate the TGF-β / Smad3 / vascular endothelial growth factor signaling pathway, and is closely related to poor prognosis. After silencing TINAGL1, the growth, proliferation, and migration of liver cancer cells can be significantly inhibited, and apoptosis of liver cancer cells can be induced, while overexpression of TINAGL1 has the opposite effect. Moreover, inhibiting TINAGL1 can also delay the growth of xenograft tumors in nude mouse models. This suggests that TINAGL1 is a potential novel biomarker for liver cancer.

[0005] In addition to being related to the occurrence and development of tumors, TINAGL1 is also related to the progression of multiple sclerosis (HK40098144A Biomarker for predicting the progression of multiple sclerosis disease) and SARS-CoV-2 infection (US20240094217A1 Method for treating SARS-COV-2 infection), but its role in the occurrence and progression of fibrosis has not been reported yet. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide the use of TINAGL1 as a direct target for screening anti-hepatic fibrosis drugs. By using TINAGL1 as a direct target, target drugs that can bind to human TINAGL1 recombinant protein can be screened out. These target drugs have been proven to be able to reduce the increase in the level of the fibrosis factor COL1A1 induced by PDGF-BB in LX-2 cells and can achieve different degrees of anti-hepatic fibrosis effects.

[0007] Meanwhile, the present invention also discloses a method for screening anti-hepatic fibrosis drugs targeting TINAGL1, anti-hepatic fibrosis drugs, and new uses of compounds.

[0008] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0009] The use of TINAGL1 as a direct target for screening anti-hepatic fibrosis drugs.

[0010] Meanwhile, the present invention also discloses a screening method for anti-hepatic fibrosis drugs, which uses TINAGL1 as a direct target to screen out target drugs; the target drugs are anti-hepatic fibrosis drugs.

[0011] In the above screening method, the method is specifically as follows: through surface plasmon resonance experiments, using human TINAGL1 recombinant protein as the binding object, target drugs that can bind to human TINAGL1 recombinant protein are screened out.

[0012] In addition, the present invention also discloses an anti-hepatic fibrosis drug, which is a drug that can bind to human TINAGL1 recombinant protein.

[0013] Among the above anti-hepatic fibrosis drugs, the drug is one or a combination of lomefloxacin, theophylline acetic acid, salvianolic acid C, bacitracin, pilocarpine nitrate, and fangchinoline.

[0014] Among the above anti-hepatic fibrosis drugs, the drug is lomefloxacin and / or theophylline acetic acid.

[0015] Finally, the present invention also discloses the application of one or a combination of lomefloxacin, theophylline acetic acid, salvianolic acid C, bacitracin, pilocarpine nitrate, and fangchinoline in the preparation of anti-hepatic fibrosis drugs.

[0016] Preferably, the application of lomefloxacin and / or theophylline acetic acid in the preparation of anti-hepatic fibrosis drugs.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] First, the present invention uses carbon tetrachloride (CCl4)-induced hepatic fibrosis mice as experimental objects, and knocks down the expression of TINAGL1 by injecting adeno-associated virus containing TINAGL1 expression interference sequence through the tail vein to explore the role of TINAGL1 in the occurrence and development of hepatic fibrosis. The results show that: knocking down TINAGL1 can significantly reduce the liver weight ratio and the content of hydroxyproline in the liver tissue of CCl4-induced hepatic fibrosis mice, and significantly relieve the pathological changes such as collagen deposition, hepatocyte granular degeneration, cell swelling, and focal inflammatory cell infiltration in the liver tissue around the intralobular and central veins. The above results indicate that: knocking down the expression of TINAGL1 can significantly improve the occurrence of hepatic fibrosis, suggesting that TINAGL1 can be used as a potential new target for the treatment of hepatic fibrosis.

[0019] Second, use biological methods to explore the mechanism of action of TINAGL1 in promoting fibrosis. The results of immunoprecipitation and surface plasmon resonance experiments show that TINAGL1 has a direct interaction with PDGF-BB.

[0020] Thirdly, using TINAGL1 as a potential drug target, 1,760 FDA-approved drugs and 640 natural products were screened by surface plasmon resonance technology and the binding specificity was verified. Six compounds (lomefloxacin, theophylline acetic acid, bacitracin, salvianolic acid C, pilocarpine nitrate, and fangchinoline) that can bind to TINAGL1 were initially obtained.

[0021] Furthermore, the anti-fibrotic effects of the screened compounds were biologically verified in a liver fibrosis cell model of human hepatic stellate cells (LX-2 cells) induced by PDGF-BB. After verification, all six compounds had anti-liver fibrosis effects to varying degrees. Among them, lomefloxacin and theophylline acetic acid could significantly reduce the elevated level of the fibrosis factor COL1A1 in LX-2 cells induced by PDGF-BB. The above results demonstrated the effectiveness of TINAGL1 as a new target for the treatment of liver fibrosis and provided a pharmacological basis for the discovery of drugs for the treatment of liver fibrosis. Description of the Drawings

[0022] Figure 1 It is the Western Blot result of TINAGL1 protein in the liver of TINAGL1 knockdown mice;

[0023] Among them, the Oil group was the group injected with olive oil (solvent control), the AAV8-Control group was the group injected with CCl4 and the empty adenovirus vector solution, and the AAV8-shTinagl1 group was the group injected with CCl4 and the specific short hairpin interfering RNA virus solution of the mouse Tinagl1 gene packaged by the adenovirus vector;

[0024] Figure 2 It is the H&E staining, Masson staining, and Sirius red staining results of the liver of TINAGL1 knockdown mice.

[0025] Figure 3 It is the result of the liver weight / body weight of TINAGL1 knockdown mice.

[0026] Figure 4 It is the result of the collagen hydroxyproline content in the liver of TINAGL1 knockdown mice.

[0027] Figure 5 It is the Western Blot result of the direct binding of PDGF-BB and TINAGL1 in 293T cells;

[0028] Figure 6 It is the kinetic curve of the binding of PDGF-BB and TINAGL1 obtained by surface plasmon resonance technology;

[0029] Figure 7AIt is the kinetic curve of the binding of TINAGL1 and lomefloxacin obtained by surface plasmon resonance technology;

[0030] Figure 7B It is the kinetic curve of the binding of TINAGL1 and theophylline acetic acid obtained by surface plasmon resonance technology;

[0031] Figure 7C It is the kinetic curve of the binding of TINAGL1 and salvianolic acid C obtained by surface plasmon resonance technology;

[0032] Figure 7D It is the kinetic curve of the binding of TINAGL1 and bacitracin obtained by surface plasmon resonance technology;

[0033] Figure 7E It is the kinetic curve of the binding of TINAGL1 and pilocarpine nitrate obtained by surface plasmon resonance technology;

[0034] Figure 7F It is the kinetic curve of the binding of TINAGL1 and fangchinoline obtained by surface plasmon resonance technology;

[0035] Figure 8 It is the graph of the intracellular content of the profibrotic factor COL1A1 mRNA detected by qRT-PCR experiment;

[0036] Figure 9 It is the structural map of the pHBAAV-U6-MCS-CMV-EGFP vector;

[0037] Figure 10 It is the structural map of the PDGF-B overexpression plasmid;

[0038] Figure 11 It is the structural map of the TINAGL1 overexpression plasmid. Specific implementation manners

[0039] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0040] Example 1

[0041] Using the adeno-associated virus AAV8 vector to specifically knockdown the expression of TINAGL1 in the liver of a CCl4-induced liver fibrosis mouse model, and injecting AAV8-Control as a control at the same time, observing the pathological changes of the mouse liver tissue to evaluate the profibrotic effect of TINAGL1.

[0042] (1) Experimental animals and feeding:

[0043] a. Experimental animals

[0044] Male C57BL / 6J mice (20 - 22 g) at 6 - 8 weeks old of SPF grade were purchased from Beijing Speywood Biotechnology Co., Ltd.

[0045] b. Animal feeding and environmental conditions

[0046] All experimental mice were housed in the SPF - grade animal house of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, with a 12 - hour light - dark cycle, and the experimental mice had free access to water and food. Before the experiment, all mice were fed adaptively for 7 days.

[0047] (2) Establishment of a CCl4 - induced liver fibrosis mouse model

[0048] Male C57BL / 6J mice at 6 - 8 weeks old were injected intraperitoneally. In the model group, they were injected with 0.75 mL / kg CCl4 (051201, Tianjin Fuchen) (dissolved in olive oil at a ratio of 1:9), and the control group mice were injected with olive oil alone, twice a week for two weeks first.

[0049] (3) Adeno - associated virus injection animal model

[0050] Recombinant adeno - associated viruses AAV8 - shControl and AAV8 - shTinagl1 were constructed and purified by Shanghai Hanheng Biotechnology Co., Ltd.

[0051] Preparation of AAV8 - shTinagl1 virus: The specific short hairpin RNA (shRNA) of the mouse Tinagl1 gene was cloned into the pHBAAV - U6 - MCS - CMV - EGFP vector (the structural diagram of this vector can be referred to Figure 9 , Figure 9 which shows the structure of the pHBAAV - U6 - MCS - CMV - EGFP vector).

[0052] The mature shRNA sequence of Tinagl1 is 5'-CCTGTTCAAGCACTCATGGAA - 3', and the nonsense control shRNA sequence is 5'-TTCTCCGAACGTGTCACGTAA - 3'.

[0053] The titer of the recombinant adeno - associated virus is 1×10 12 viral genomes / mL.

[0054] Knockdown of TINAGL1 expression in mice: C57BL / 6J mice that had been modeled with CCl4 for two weeks were injected via the tail vein with AAV8-shControl or AAV8-shTinagl1, 100 μL per mouse. Then, CCl4 injection was carried out for 6 weeks, and subsequently, serum and liver tissue samples were collected.

[0055] (4) Terminal liver tissue sampling

[0056] Sampling was carried out 48 h after the last CCl4 injection. After weighing the mice, the livers were quickly removed, placed on weighing paper, and the residual blood on the surface was wiped off. Then, they were photographed and weighed. At the same time, a small part of the liver was cut and fixed in 4% paraformaldehyde fixative, and serum samples and liver tissues were collected and stored in an -80 °C refrigerator for subsequent analysis.

[0057] (5) Related analysis of pathological staining

[0058] a. Liver dehydration:

[0059] The liver tissue was fixed in 4% paraformaldehyde fixative for 24 h, dehydrated with different concentrations of ethanol and pretreated with xylene in the following order:

[0060] Soak in 75% ethanol solution overnight; then soak in 85% ethanol for 2 h; soak in 95% ethanol for 2 h and repeat this step twice; then soak in absolute ethanol for 1.5 h; soak in absolute ethanol for 1 h; soak in a 1:1 mixture of xylene and absolute ethanol for 20 min; then soak in xylene for 10 min and repeat this step twice;

[0061] b. Embedding and sectioning: The flat liver tissue was embedded in paraffin, and sections were cut to a thickness of about 5 μm with a microtome;

[0062] c. Dewaxing and rehydration of sections: The paraffin sections of liver tissue were placed in a 65 °C oven for 30 min; soaked in xylene for 10 min; soaked in xylene again for 5 min and repeat this step twice; then placed in absolute ethanol for 5 min; 95% ethanol for 5 min; 85% ethanol for 5 min; 75% ethanol for 5 min; and finally soaked in double-distilled water for 5 min;

[0063] d. H&E staining: Immerse the sections in hematoxylin solution for 5 min; then rinse with tap water for 5 min; differentiate with 0.5% hydrochloric acid alcohol for 10 s; then rinse with tap water until the sections turn blue; immerse in 70% ethanol for 5 min; 80% ethanol for 5 min; stain with eosin solution for 5 s; then immerse in 95% ethanol for 1 min; immerse in 95% ethanol again for 5 min; absolute ethanol for 1 min, and absolute ethanol again for 5 min; then immerse in a 1:1 mixture of xylene and absolute ethanol for 5 min; immerse in xylene for 1 min; xylene again for 5 min; air dry and mount the coverslip.

[0064] e. Sirius red staining: Slightly drain the water from the sections and immerse them in Weigert iron hematoxylin staining solution for 2 min; rinse the sections through two tanks of tap water and immerse them in the modified Sirius red staining solution for 30 min; then immerse the sections in four tanks of absolute ethanol for 3 - 5 s each in sequence; quickly rinse and dehydrate, then put them into clean xylene for clearing for 1 min, and mount with neutral gum.

[0065] f. Masson staining: Dewax the paraffin sections to water; then immerse the sections in Regaud hematoxylin staining solution for 5 - 10 min and rinse with running tap water; differentiate for several seconds with the differentiating solution and wash with tap water; immerse the sections in 0.1 - 1% lithium carbonate for 5 min to increase the bluing degree and wash with tap water; immerse the sections in Masson ponceau acid fuchsin solution for 6 min and rinse with tap water; immerse in 2% glacial acetic acid aqueous solution for 1 min; without washing, slightly drain and directly immerse in 1% phosphomolybdic acid aqueous solution for differentiation for 3 - 5 min; rinse and differentiate the sections with 1% acetic acid, dehydrate with two tanks of absolute ethanol; immerse the sections in the third tank of absolute ethanol for 5 min, then immerse in xylene for 5 min until clear, and mount with neutral gum; examine under a microscope and collect and analyze the images.

[0066] (6) Determination of hydroxyproline content in the liver

[0067] a. Sample hydrolysis: Accurately weigh 100 mg of the liver tissue to be measured and put it into a test tube, add 1 mL of hydrolysis solution and mix well. After covering, hydrolyze in a boiling water bath for 20 min (mix once every 10 min during this period to make the hydrolysis more complete).

[0068] b. Adjust the pH value to around 6.0 - 6.8: After cooling each test tube under running water, add 10 μL of indicator and shake well; then add 1 mL of pH adjustment solution A and mix evenly (the solution is red at this time); carefully add pH adjustment solution B drop by drop to each tube, and mix well after each drop is added until the color of the indicator in the liquid changes to yellowish green, at which time the pH value is around 6.0 - 6.8; then add double-distilled water to 10 mL and mix well; finally, take 3 - 4 mL of the diluted hydrolyzate, add an appropriate amount of activated carbon (about 20 - 30 mg or so, based on the supernatant being clear and colorless after centrifugation), mix well, centrifuge at 3500 rpm for 10 min, and carefully take 1 mL of the supernatant for detection.

[0069] c. Detection: Take 1 mL of the supernatant to be tested and the standard application solution, and at the same time set distilled water as the blank control. Then add 0.5 mL of reagent one, mix well and let stand for 10 min; then add 0.5 mL of reagent two, mix well and let stand for 5 min; finally, add 0.5 mL of reagent three, mix well, incubate in a water bath at 60 °C for 15 min, cool, centrifuge at 3500 rpm for 10 min, take 200 μL of the supernatant and place it in a 96-well plate, and measure the OD value of each well with an enzyme-linked immunosorbent assay (ELISA) reader at 510 nm.

[0070] d. The hydroxyproline content is calculated according to the following formula:

[0071]

[0072] As Figure 1 shown, the expression of TINAGL1 in the liver of mice in the TINAGL1 knockdown group was significantly decreased ( Figure 1 ); and it could significantly relieve pathological changes such as granular degeneration of hepatocytes, cell swelling, focal inflammatory cell infiltration in the lobule and around the central vein, and collagen deposition in the liver tissue ( Figure 2 ). At the same time, the ratio of liver weight to body weight ( Figure 3 ) and the content of collagen hydroxyproline in the liver tissue ( Figure 4 ) of mice in the TINAGL1 knockdown group could be significantly reduced in CCl4-induced liver fibrosis mice. The above results indicate that knocking down the expression of TINAGL1 can significantly improve the occurrence of liver fibrosis, suggesting that TINAGL1 can be used as a potential new target for the treatment of liver fibrosis.

[0073] Example 2

[0074] Explore the interaction relationship between TINAGL1 and PDGF-BB in vitro using co-immunoprecipitation to clarify the mechanism of action of TINAGL1 in promoting fibrosis.

[0075] (1) Co-immunoprecipitation experiment

[0076] a. 293T cells were seeded at 3×10 4 cells / cm 2Plated in a T75 cell culture flask;

[0077] b. After culturing the cells overnight, the Huh7.5 cells were divided into three groups and transfected with 20 μg of the TINAGL1 overexpression plasmid (product number JY6187, Hunan Fenghui Biotechnology Co., Ltd.), 20 μg of the PDGF-B overexpression plasmid (product number HG10572, Beijing Proteintech Group Co., Ltd.), or co-transfected with 10 μg of the PDGF-B plasmid and 10 μg of the TINAGL1 plasmid. The plasmid maps of TINAGL1 and PDGF-B are shown in Figure 10 and Figure 11 ; Figure 10 is the structural map of the PDGF-B overexpression plasmid; Figure 11 is the structural map of the TINAGL1 overexpression plasmid.

[0078] c. After 48 h, the medium was discarded, and the cells were collected. The cells were rinsed once with pre-cooled PBS, and 1 mL of IP lysis buffer (protease inhibitor was added in advance) was added to lyse the cells and extract the proteins. Subsequently, BCA quantification was performed to adjust to a uniform loading amount; at the same time, the magnetic beads were equilibrated to room temperature, and the magnetic beads were mixed by inverting up and down or gently vortexing (they can be placed on a turntable and rotated);

[0079] d. 200 μL of the Flag magnetic bead mixture (A36797, ThermoFisher) or 100 μL of the HA magnetic bead mixture (88838, ThermoFisher) was added to a 1.5 mL centrifuge tube, and 500 μL of IP lysis buffer was added and gently vortexed;

[0080] e. The centrifuge tube was placed on a magnetic stand, and the supernatant was discarded;

[0081] f. 500 μL of IP lysis buffer was added, gently vortexed, placed on a magnetic stand, and the supernatant was discarded;

[0082] g. The above steps were repeated;

[0083] h. 0.8 mL of cell lysate was added to the magnetic beads (100 μL of the sample was reserved as Input, and 6×loading buffer was added simultaneously), and gently vortexed; the sample was incubated overnight at 4°C with rotation;

[0084] i. The magnetic beads were collected on a magnetic stand, and the supernatant was retained as Output (the volume was the same as that of the input, and 6×loading buffer was added simultaneously);

[0085] g. 1 mL of IP lysis buffer was added, gently vortexed multiple times, and washed at least three times using a magnetic stand;

[0086] k. Add 100 μL of 1× Lane Marker Reducing sample buffer (Thermo 39000), boil the sample and vortex it, then place it on a magnetic stand. The supernatant is the precipitated protein sample, and then SDS-PAGE electrophoresis (60V electrophoresis) is performed.

[0087] l. The transfer voltage is 106V. After 60 minutes, block it with BSA solution at room temperature for 1 hour.

[0088] m. Incubate with the primary antibody overnight at 4°C.

[0089] n. Incubate with the secondary antibody: Wash three times with TBST, 10 minutes each time. Use a secondary antibody that does not show heavy and light chains (001-100-005; 025-100-005, Critical Point Biotechnology). Incubate at room temperature for 1 hour. After incubation, wash three times with TBST.

[0090] o. Develop the image: Develop the image with ECL chemiluminescence solution and use a Bio-Rad imaging analysis system.

[0091] (2) Surface plasmon resonance experiment

[0092] Use a low-density SR7000 GOLD SENSOR SLIDE NINTA surface chip (13206063, Reichert) to perform kinetic detection on a Reichert 4SPR instrument.

[0093] Fix 60 μg of human TINAGL1 recombinant protein (with a histidine tag at the C-terminus, product number CB22, Suzhou Novoprotein Science & Technology Co., Ltd.) in the channels of the NINTA surface chip with 40 mM nickel sulfate (656895, Sigma-Aldrich). Dissolve human PDGF-BB recombinant protein (product number 100-14B, Peprotech) in PBST and inject the PDGF-BB recombinant protein solution diluted into different concentration gradients into the flow system at a flow rate of 62.5 μL / min. At the same time, set a solvent control (PBST) and a blank control (a chip without fixed protein). Set the binding time to 4 minutes and the dissociation time to 5 minutes. All solutions need to be filtered and sterilized through a 0.22 μm pore size filter and degassed at room temperature for 30 minutes. All operation steps are carried out according to the operation instructions of the Reichert 4SPR instrument.

[0094] After the experiment, use TraceDrawer V1.8.1 software to analyze the kinetic detection results, calculate the equilibrium dissociation constant (K D ), and analyze whether there is an interaction between TINAGL1 and PDGF-BB proteins.

[0095] In this example, the tagged TINAGL1-Flag and PDGF-BB-HA plasmids were transfected into HEK293T cells and Huh7.5 cells, and the expression of related proteins was detected after direct immunoprecipitation with Flag and HA antibodies respectively. The results showed that there was an interaction between TINAGL1 and PDGF-BB in HEK293T cells and Huh7.5 cells ( Figure 5 ). The surface plasmon resonance results showed that TINAGL1 and PDGF-BB had a direct interaction, and its equilibrium dissociation constant K D value was about 0.185 μM. Usually, a K D value in the μM level is considered a medium-strength binding. Therefore, the experimental results showed that there was a medium-strength direct interaction between TINAGL1 and PDGF-BB proteins ( Figure 6 ).

[0096] Example 3

[0097] Using TINAGL1 as a potential drug target, novel anti-hepatic fibrosis drugs that may bind to TINAGL1 were screened from the FDA-approved drug library and natural product library by surface plasmon resonance experiments

[0098] The experimental method was the same as before. A low-density SR7000 GOLD SENSOR SLIDE NINTA chip was used for kinetic detection on a Reichert4SPR instrument. 60 μg of human TINAGL1 recombinant protein was immobilized in the channels of the NINTA surface chip with 40 mM nickel sulfate. The diluted solutions of FDA-approved drugs and natural products with an initial concentration of 10 μM were injected into the flow system at a flow rate of 62.5 μL / min, and a 1% DMSO PBST solution was used as a blank control. The binding time was set to 4 min and the dissociation time was set to 5 min. For drugs that may bind, further rescreening was carried out according to the concentration gradient of 50 μM / 25 μM / 12.5 μM / 6.25 μM / 3.125 μM / 1.5625 μM.

[0099] In this example, 2400 compounds were screened, such as: polydatin, shikimic acid, vinblastine, sinomenine hydrochloride, digitonin, arecoline, methyl cholate, rotenone, alizarin, paulownic acid, lipoamide, cimetidine, triamcinolone, betamipron, dasatinib, candesartan, ramelteon, nadifloxacin, drospirenone, tenofovir, methacholine chloride, tipiracil hydrochloride, lomefloxacin, theophylline acetic acid, salvianolic acid C, bacitracin, pilocarpine nitrate, and fangchinoline, etc. (Due to space limitations, the types of compounds cannot be listed one by one);

[0100] Finally, six compounds with definite binding ability were screened out: lomefloxacin, theophylline acetic acid, salvianolic acid C, bacitracin, pilocarpine nitrate, and fangchinoline.

[0101] Example 4

[0102] Verification of the anti-hepatic fibrosis effect of candidate compounds

[0103] (1) Cell line

[0104] The human immortalized hepatic stellate cell line LX-2 was routinely cultured and passaged with DMEM + GlutaMAXTM-I medium containing 10% inactivated fetal bovine serum and 100 U / mL penicillin / streptomycin.

[0105] (2) Verification of the efficacy of candidate compounds

[0106] LX-2 cells were seeded in 96-well plates and starved with serum-free medium 36 h later. After 24 h of cell starvation, 20 ng / mL of PDGF-BB recombinant protein and 10 μM of the candidate compound were added. At the same time, blank control wells were set up. After 48 h, the cells were lysed and total RNA was extracted. According to the kit instructions, one-step qRT-PCR was used with GAPDH as the internal reference to detect the expression of the fibrosis gene COL1A1 mRNA.

[0107] Using TINAGL1 as a potential drug target, 1760 FDA-approved drugs and 640 natural products were screened by high-throughput screening and binding specificity verification using surface plasmon resonance technology, as Figure 7A shown in -F. Initially, 6 compounds (lomefloxacin, theophylline acetic acid, bacitracin, salvianolic acid C, pilocarpine nitrate, and fangchinoline) that could bind to TINAGL1 were obtained. Further, the anti-fibrosis effects of the screened compounds were biologically verified in a PDGF-BB-induced human hepatic stellate cell (LX-2 cell) hepatic fibrosis cell model. After verification, all 6 compounds had anti-hepatic fibrosis effects to varying degrees. Among them, lomefloxacin and theophylline acetic acid could significantly reduce the content of the fibrosis factor COL1A1 mRNA in LX-2 cells induced by PDGF-BB. The above results demonstrated the effectiveness of TINAGL1 as a new target for the treatment of hepatic fibrosis and provided a pharmacological basis for the discovery of drugs for the treatment of hepatic fibrosis ( Figure 8 ).

[0108] The applicant declares that the present invention illustrates the process method of the present invention through the above embodiments, but the present invention is not limited to the above process steps, which does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of the raw materials selected for the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Use of lomefloxacin and / or theophylline acetic acid in the preparation of anti-liver fibrosis drugs; the anti-liver fibrosis drugs are LX-2 cell fibrosis factors used to reduce PDGF-BB-induced human hepatic stellate cell liver fibrosis cell model COL1A1 mRNA content of the drug.

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