Application of riboflavin kinase inhibitor in preparation of medicine for preventing and treating liver injury fibrosis

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of a riboflavin kinase inhibitor in preparation of a medicine for preventing and treating liver injury fibrosis. According to the application of the riboflavin kinase inhibitor in preparing the medicine for preventing and / or treating liver injury fibrosis, the riboflavin kinase inhibitor can reprogram energy metabolism by inhibiting the catalytic activity of iNOS and the generation of NO and regulate polarization of macrophages, so that the effects of promoting polarization of macrophages M (IL-4) and promoting liver injury fibrosis are achieved. And M (IFN-gamma) polarization is inhibited. The invention not only discloses a new role of RFK-mediated riboflavin metabolism in macrophage polarization, but also defines that interference of RFK-iNOS interaction or operation of riboflavin metabolism can be used as a strategy for treating macrophage-mediated liver injury fibrosis.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of riboflavin kinase inhibitors in the preparation of drugs for preventing and treating liver damage fibrosis. Background Art

[0002] Fibrosis refers to the pathological process in which the fibrous connective tissue increases and the parenchymal cells decrease in organ tissues. In this process, the extracellular matrix (mainly composed of collagen, proteoglycans, glycosaminoglycans, glycoproteins and elastin) increases abnormally and deposits excessively, leading to the gradual destruction of organ structure, functional impairment, and even failure. Fibrosis can occur in a variety of organs, such as the liver, lungs, kidneys, heart, etc.

[0003] Liver fibrosis is a process in which liver cells swell, degenerate, and necrotize, accompanied by the proliferation of fibrous tissue, after the liver is damaged by various factors (such as viruses, alcohol, drugs, fat deposition, etc.). This process is actually a scar repair response of the liver to damage, but excessive repair can lead to structural damage and functional impairment of the liver. Over time, the proliferation and deposition of fibrous tissue will gradually worsen, eventually leading to the occurrence of liver fibrosis. Liver fibrosis is an important risk factor for liver cancer. Long-term liver damage and fibrosis can lead to mutation and malignant transformation of liver cells, thereby increasing the risk of liver cancer.

[0004] As a repair response of the liver to injury, liver fibrosis is often accompanied by the participation of the immune system in its occurrence and development. Specifically, the immune response can promote the occurrence and development of liver fibrosis. When the liver is damaged, the immune system will identify and remove damaged liver cells, while releasing a large number of inflammatory mediators and cytokines. These inflammatory mediators and cytokines will not only aggravate the inflammatory response of the liver, but also promote the proliferation and deposition of fibrous tissue, thereby accelerating the process of liver fibrosis. In addition, inflammation is one of the important driving factors of liver injury fibrosis. Inflammatory response of the liver caused by any reason may cause damage and death of liver cells, thereby triggering the repair mechanism of the liver. During the repair process, fibrous tissue will proliferate and deposit in the damaged area to replace the damaged liver tissue. Over time, the proliferation and deposition of this fibrous tissue will gradually increase, eventually leading to the occurrence of liver fibrosis. Immunity and inflammation are intertwined and influence each other in the occurrence and development of liver injury fibrosis. On the one hand, the immune response can trigger the inflammatory response of the liver, leading to damage and death of liver cells; on the other hand, the inflammatory response can further activate the immune system, forming a vicious circle. This interaction not only exacerbates hepatocyte damage and fibrosis, but may also lead to severe damage to liver function and progression of the disease.

[0005] Alternatively activated macrophages (M2, anti-inflammatory) promote the reversal of liver fibrosis by:

[0006] (1) Enhance the expression of matrix metalloproteinases (MMPs) such as MMP-1 and MMP-13, and regulate the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs), leading to the degradation of the extracellular matrix. (2) Macrophage-specific ARG1 acts as an inhibitor of CD4+T cell responses, inhibiting Th2-dependent inflammation and fibrosis. (3) Phagocytosis of damaged hepatocytes. (4) Inducing senescence and apoptosis of activated hepatic stellate cells (HSC). Macrophage polarization is an important regulatory mechanism for the body to respond to different microenvironmental stimuli. It plays a vital role in maintaining homeostasis, defending against pathogens, regulating inflammation, repairing tissues, and participating in immune responses. By developing drugs that can promote macrophage polarization, precise treatment can be carried out for specific diseases such as tumors, infections, and autoimmune diseases. For example, the development of drugs that can induce macrophage polarization to M2 has the potential to prevent and treat liver injury and fibrosis. Summary of the invention

[0007] In order to solve the above problems, the present invention provides the use of riboflavin kinase inhibitors in the preparation of drugs for preventing and / or treating liver injury fibrosis, wherein the riboflavin kinase inhibitors can inhibit the catalytic activity of iNOS and the production of NO to reprogram energy metabolism and regulate macrophage polarization, thereby promoting macrophage M (IL-4) polarization and inhibiting M (IFN-γ) polarization. The present invention not only reveals the new role of RFK-mediated riboflavin metabolism in macrophage polarization, but also clarifies that interfering with RFK-iNOS interaction or manipulating riboflavin metabolism can be used as a strategy for treating macrophage-mediated liver injury fibrosis.

[0008] In a first aspect, the present invention provides the use of a riboflavin kinase inhibitor in the preparation of a drug for preventing and / or treating liver fibrosis.

[0009] Specifically, the riboflavin kinase inhibitor modulates macrophage polarization.

[0010] More specifically, the riboflavin kinase inhibitor reprograms energy metabolism and regulates macrophage polarization by inhibiting the catalytic activity of iNOS and the production of NO.

[0011] Preferably, the macrophage polarization inhibits M1 type but promotes M2 type polarization.

[0012] Specifically, the liver injury fibrosis includes, but is not limited to, fibrosis caused by viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver injury or autoimmune liver disease.

[0013] Specifically, the riboflavin kinase inhibitor is the sole or main active ingredient of the drug.

[0014] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0015] More specifically, the pharmaceutically acceptable excipient is one or more of a filler, a binder, a disintegrant, a lubricant, a stabilizer, a surfactant, an antioxidant, a pH adjuster or a preservative.

[0016] Preferably, the pharmaceutically acceptable excipient is at least one of lactose, mannose, mannitol, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, mineral oil, vitamin C, vitamin E, citric acid, phosphoric acid, sodium benzoate, potassium sorbate, Tween-80, poloxamer, glutamic acid and glycine.

[0017] Further preferably, when preparing a drug containing a riboflavin kinase inhibitor, the type and amount of excipients are precisely controlled according to the characteristics and application requirements of the specific drug.

[0018] Specifically, the dosage form of the drug includes, but is not limited to, injection, tablet, capsule, granule, powder, cream, ointment, gel or spray.

[0019] Further specifically, the injection may be in the form of a solution, suspension or emulsion;

[0020] The tablets may be ordinary tablets, enteric-coated tablets, sustained-release tablets or controlled-release tablets;

[0021] The capsule may be a hard capsule or a soft capsule, and the contents may be powder, granules or pellets;

[0022] The granules may be in the form of fast dissolving or slow dissolving;

[0023] The powder may be a form of pharmaceutical powder that can be used for topical treatment or as an alternative to oral medication.

[0024] Specifically, the drug is administered by a method selected from oral, intravenous, topical, intradermal or subcutaneous injection.

[0025] Specifically, the drug can also be used in combination with other drugs for preventing and / or treating liver fibrosis.

[0026] More specifically, the other drugs for preventing and / or treating liver fibrosis are colchicine, silymarin, obeticholic acid, polyene phosphatidylcholine, entecavir, tenofovir, interferon, adefovir, lamivudine, Anluo Huaxian Pills, Fuzheng Huayu Capsules, Fufang Biejia Soft Liver Tablets, Xiao Chaihu Decoction, Qiangan Capsules or Dahuang Zhechong Pills.

[0027] More specifically, the combined use is simultaneous use or sequential use.

[0028] Specifically, the dosage of the drug is adjusted according to the severity of the disease, the age of the patient, the method of administration and the course of treatment.

[0029] The technical effects achieved by the present invention are:

[0030] The present invention provides the use of a riboflavin kinase inhibitor in the preparation of a drug for preventing and / or treating liver injury fibrosis. The riboflavin kinase inhibitor can inhibit the catalytic activity of iNOS and the production of NO to reprogram energy metabolism and regulate macrophage polarization, thereby promoting macrophage M (IL-4) polarization and inhibiting M (IFN-γ) polarization. The present invention not only reveals the new role of RFK-mediated riboflavin metabolism in macrophage polarization, but also clarifies that interfering with RFK-iNOS interaction or manipulating riboflavin metabolism can be used as a strategy for treating macrophage-mediated liver injury fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Diagram of the signaling pathway of riboflavin kinase inhibitors on macrophage polarization.

[0032] Figure 2 RFK promotes the polarization of M (IFN-γ) but inhibits the polarization of M (IL-4) through its enzymatic activity; AC in the figure represents the effect of RFK deficiency on macrophage M1-related marker genes; DF represents the effect of RFK deficiency on macrophage M2-related marker genes; GJ represents the effect of RFK enzymatic activity on macrophage polarization.

[0033] Figure 3 Riboflavin metabolism inhibits macrophage M2 polarization in CCl4-induced liver fibrosis model; A in the figure represents the effect of RFK deficiency on AST and ALT in mouse serum; BC represents the effect of RFK deficiency on tissue liver damage; D represents the effect of RFK deficiency on macrophage M2-related marker genes in liver tissue.

[0034] Figure 4 RFK regulates macrophage polarization through FAD; AB in the figure represents the effect of FAD on the expression of macrophage M1-related marker genes caused by RFK deficiency; CD represents the effect of FAD on the expression of macrophage M2-related marker genes caused by RFK deficiency.

[0035] Figure 5RFK regulates macrophage polarization through NO; A in the figure represents the investigation of whether the macrophage polarization phenotype caused by RFK deficiency is caused by ROS; BF represents the effect of NO agonist on the expression of macrophage M1 / M2 related marker genes caused by RFK deficiency; GJ represents the effect of NO inhibitor on the expression of macrophage M1 / M2 related marker genes caused by RFK deficiency.

[0036] Figure 6 RFK deficiency regulates energy metabolism through NO-mediated tricarboxylic acid cycle and mitochondrial electron transport chain remodeling; AD in the figure represents the effect of NO agonist on ECAR / OCR of macrophages caused by RFK deficiency; EH represents the effect of NO agonist on glycolysis, oxidative phosphorylation-related metabolic enzyme activities and products of macrophages caused by RFK deficiency; IJ represents the effect of NO agonist on mitochondrial complex activity caused by RFK deficiency.

[0037] Figure 7 RFK deficiency regulates energy metabolism through NO-mediated remodeling of the tricarboxylic acid cycle and mitochondrial electron transport chain; AH in the figure represent the effects of glycolysis / oxidative phosphorylation (OM / 2-DG) on macrophage energy metabolism caused by RFK deficiency; I represents the effects of NO agonist and RFK deficiency on HIF-1α.

[0038] Figure 8 RFK interacts with iNOS and promotes iNOS activity; AD in the figure represents that RFK can interact with iNOS; E represents that RFK mainly interacts with the iNOS-1-500aa domain; FG represents that iNOS mainly interacts with the RFK-60-90aa domain; HL represents the influence of RFK S64 and Y91 sites on the interaction between RFK and iNOS, iNOS enzyme activity, and the expression of macrophage M1 / M2 related marker genes.

[0039] Fig. 9 The interaction between RFK and iNOS is crucial for iNOS enzyme activity, subsequent metabolic reorganization and regulation of macrophage polarization; in the figure, A represents the ClusPro protein docking results of RFK and iNOS; B represents the mass spectrometry results of proteins interacting with RFK; CD represents that IFN-γ stimulation promotes the interaction between RFK and iNOS; EF represents that IL-4 stimulation has no effect on the interaction between RFK and iNOS; G represents the schematic diagram of the iNOS model structure; HI represents that RFK mainly interacts with the iNOS-1-500aa domain; JK represents the Consurf analysis results of RFK and the schematic diagram of the S64 and Y91 sites of RFK; L represents the effect of the S64 and Y91 sites of RFK on the production of NO. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. M (IFN-γ) polarization in the present invention is also referred to as classical activation or M1 polarization; M (IL-4) polarization is also referred to as alternative activation or M2 polarization.

[0041] Example 1 Animals, Materials and Statistical Analysis Used in the Present Invention

[0042] 1. Animals

[0043] R fl / fl The mice were generated by Saiye Bio (Guangzhou, China) using CRISPR-Cas9 technology. fl / fl Mice were mated with Lyz2-Cre mice to generate myeloid cell-specific RFK knockout mice. fl / fl Lyz2-Cre + (called Rfk fl / fl Lyz2-Cre- Mice). They were used as animals in the examples of the present invention.

[0044] PHGDH fl / fl The mice were generated by Saiye Bio (Guangzhou, China) using CRISPR-Cas9 technology. fl / fl Mice were mated with Lyz2-Cre mice to generate myeloid cell-specific PHGDH knockout mice. fl / fl Lyz2-Cre + (called PHGDH in mice and their littermates fl / fl Lyz2-Cre- Mice). The animals were used as the comparative example 1 of the present invention.

[0045] ADPG fl / fl The mice were generated by Saiye Bio (Guangzhou, China) using CRISPR-Cas9 technology. fl / fl Mice were mated with Lyz2-Cre mice to generate myeloid cell-specific ADPG knockout mice. fl / fl Lyz2-Cre + (called ADPG in mice and their littermates fl / fl Lyz2-Cre- Mice). Used as comparative example animals of the present invention. Used as comparative example 2 animals of the present invention.

[0046] All mice were of C57BL / 6 background. Mice were housed in individually ventilated cages in a temperature- and light-controlled SPF facility and provided with unlimited food and water.

[0047] 2. Cell lines and primary cell culture

[0048] RAW264.7 cells (ATCC, TIB-71) were cultured at 37°C and 5% CO2 in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). HEK293T (ATCC, CRL-3216) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 1% PS. To isolate mouse peritoneal macrophages, each mouse was injected intraperitoneally with 1 mL of 3% Brewer's collagenase medium. On day 4, mice were sacrificed and injected intraperitoneally with 8 mL of PBS buffer precooled to 4°C and supplemented with 3% FBS. Peritoneal fluid was slowly drawn and centrifuged, and erythrocytes were lysed with erythrocyte lysis buffer (Solarbio) and resuspended in RPMI-1640 containing 10% FBS. Bone marrow derived macrophages (BMDMs) were isolated from the tibia and femur of mice and cultured in RPMI-1640 medium containing 10% FBS and 30% L929 cell supernatant at 37°C and 5% CO2 for 7 days. For macrophage polarization experiments, macrophages were treated with 100 ng / mL IFN-γ (50709-MNAH, Sino Biological), 100 ng / mL IFN-γ + 100 ng / mL LPS (L4516, Sigma), or 20 ng / ml IL-4 (404-ML, R&D Systems), and the treatment time was carried out according to the experimental requirements.

[0049] 3. Statistical analysis

[0050] Sample size and significance level are indicated in the legend. Statistical significance was determined by a two-tailed Student's t test. For analysis of differences between more than two groups, one-way analysis of variance (ANOVA) was used. Fluorescence imaging was performed in a blinded state. The mean ± standard error (SEM) of all quantitative data is from at least three independent experiments. Data were analyzed using GraphPad Prism software (GraphPad Software). Significance levels less than 0.05 were considered statistically significant (*p<0.05, **p<0.01, ***p<0.001). Results with p values ​​of 0.05 or greater were considered not significant (NS).

[0051] Example 2 Effect of riboflavin kinase on liver fibrosis

[0052] 1. Experimental methods

[0053] 1.1 RNA extraction and RT-PCR analysis

[0054] RNA was extracted using TRIZOL reagent (#P118-05, Genstar) and subsequently reverse transcribed using a reverse transcription system (#A236-04, Genstar). qPCR analysis was then performed using SYBR Green PCR Mix (#B21203, Bimake) and designed primers in an ABI 7500 detection system. The data were normalized according to the expression level of the internal reference gene β-actin for each sample. The qPCR primers used are as follows:

[0055] Rfk forward 5′-CCCCACAGCCAATTTTCCTGA-3′ (SEQ ID NO. 1),

[0056] Rfk reverse 5′-TGCTCACCACCATTTTATGGAC-3′ (SEQ ID NO. 2);

[0057] Cxcl9 forward 5′-TCCTTTTGGGCATCATCTTCC-3′ (SEQ ID NO. 3),

[0058] Cxcl9 reverse 5′-TTTGTAGTGGATCGTGCCTCG-3′ (SEQ ID NO. 4);

[0059] Nos2 forward 5′-GAAACGCTTCACTTCCAATG-3′ (SEQ ID NO. 5),

[0060] Nos2 reverse 5′-AATCCACAACTCGCTCCAA-3′ (SEQ ID NO. 6);

[0061] Il-6 forward 5′-TTGCCTTCTTGGGACTGAT-3′ (SEQ ID NO. 7),

[0062] Il-6 reverse 5′-TTGCCATTGCACAACTCTT-3′ (SEQ ID NO. 8);

[0063] Il-1β forward 5′-GAAATGCCACCTTTTGACAGTG-3′ (SEQ ID NO. 9),

[0064] Il-1β reverse 5′-TGGATGCTCTCATCAGGACAG-3′ (SEQ ID NO. 10);

[0065] Tnf forward 5′-CCTCTTCTCATTCCTGCTTG-3′ (SEQ ID NO. 11),

[0066] Tnf reverse 5′-GTCACCCCGTCCACATCTT-3′ (SEQ ID NO. 12);

[0067] Arg1 forward 5′-CCACAGTCTGGCAGTTGGAAG-3′ (SEQ ID NO. 13),

[0068] Arg1 reverse 5′-GGTTGTCAGGGGAGTGTTGATG-3′ (SEQ ID NO. 14);

[0069] Mgl1 forward 5′-CAGAATCGCTTAGCCAATGTGG-3′ (SEQ ID NO. 15),

[0070] Mgl1 reverse 5′-TCCCAGTCCGTGTCCGAAC-3′ (SEQ ID NO. 16);

[0071] Mgl2 forward 5′-TTCAAGAATTGGAGGCCACT-3′ (SEQ ID NO. 17),

[0072] Mgl2 reverse 5′-CAGACATCGTCATTCCAACG-3′ (SEQ ID NO. 18);

[0073] Retnla forward 5′-CCAATCCAGCTAACTATCCCTCC-3′ (SEQ ID NO. 19),

[0074] Retnla reverse 5′-CACTTGGTGGTTTGCTACG-3′ (SEQ ID NO. 20).

[0075] 1.2 Protein extraction and immunoblotting analysis

[0076] Cells were lysed using radioimmunoprecipitation assay lysis buffer supplemented with a protease inhibitor cocktail. Proteins were separated by SDS-PAGE for western blot analysis. Target protein levels were analyzed using specific antibodies. Visualization was performed using an ECL chemiluminescent detection kit (Millipore). The antibodies used were as follows: RFK (1:1000; #15813-1-AP, Proteintech); iNOS (1:1000;

[0077] #13120S, CST); ARG1 (1:1000; #93668S, CST); β-actin (1:1000; #sc-47778, SantaCruz); Tubulin (1:1000; #ab7792, Abcam); FLAG (1:1000; #20543-1-AP, Proteintech); IL-1β (1:1000; #ab308765, Abcam); GST (1:1000; #10000-0-AP, Proteintech); His (1:1000; #AH367, Beyotime); HA (1:1000; #3724, CST).

[0078] 1.3 Flow cytometry

[0079] Peritoneal cells were extracted as single cell suspensions and stained with specific surface antibodies in a staining buffer containing 2% BSA. The cells were then washed three times and incubated with anti-CD45-PE (#103105, Biolegend), anti-CD170-FITC (#155504, Biolegend), anti-F4 / 80-PE-Cy5 (#123112, Biolegend) and anti-CD11b-APC (#101211, Biolegend) antibodies, and then flow cytometry was performed using a BD Caliber flow cytometer (BD Biosciences). Data were analyzed by FlowJo (Tree Star) software.

[0080] 1.4 Enzyme-linked immunosorbent assay (ELISA)

[0081] The peritoneal macrophage supernatant was collected and measured using the indicated ELISA kits. IL-6 (#EK0411, Boster); IL-1β (#EK0394, Boster); TNF-α (#EK0527, Boster); CCL17 (#EK0682, Boster); CCL22 (#EK0447, Boster); The detection was performed according to the reagent manufacturer's protocol.

[0082] 1.5 Plasmids and transfection

[0083] Expression plasmids for RFK and iNOS were constructed using standard molecular biology techniques and cloned into the pLenti-3Flag / HA vector. Point mutations (RFK / iNOS) were generated by site-directed mutagenesis; plasmids encoding wild-type RFK or iNOS protein were used as templates. All RFK and iNOS mutant constructs were generated using Phusion Hot Start II DNA Polymerase (ThermoFisher Scientific). Plasmids with different domains of iNOS (1-500 / 1-970 / 501-1144 / 971-1144) were generated based on pLenti-iNOS-HA. pGEX-4T-1-GST-RFK was generated by cloning RFK cDNA into the pGEX-4T-1-GST vector, which was linearized with EcoRI and Xho1 (Thermo Fisher Scientific). pET28a-His-iNOS was generated by cloning the iNOS cDNA fragment into the pET28a-His vector, which was linearized by EcoRI (Thermo Fisher Scientific). All generated plasmids and mutations were confirmed by sequencing. Appropriate plasmids and siRNAs were transfected into RAW264.7 cells, HEK293T cells, and primary macrophages using Lipofectamine 2000 / RNAiMAX (Invitrogen). The siRNA sequence is as follows: siRfk: 5′-CCAGGCACTTAAACATAATTT-3′ (SEQ ID NO.21). The reagents used were as follows: FAD (#HY-B1654, MCE); DETA NONOate (#HY-136278, MCE); guanidine hydrochloride aminoguanidine (#HY-B1041, MCE); NAC (#HY-B0215, MCE).

[0084] 1.6CCl4-induced liver fibrosis model

[0085] Liver fibrosis was induced by intraperitoneal injection of CCl4 (1.0 mL / kg; #C27710, Acmec Biochemical), diluted in corn oil at a ratio of 1:3, 3 times a week for 4 weeks (a total of 12 injections). Serum alanine aminotransferase (ALT) (#C009-2-1) and aspartate aminotransferase (AST) (#C010-2-1) were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute). For histological analysis, the liver was dissected and fixed in 4% paraformaldehyde solution overnight, and then stained with hematoxylin-eosin for routine examination or sirius red staining (#G1471, Solarbio) to visualize fibrotic deposition. The fibrotic area was quantified by ImageJ software (NIH). Liver sections embedded in OCT were subjected to immunofluorescence staining. Liver RNA was extracted for qPCR detection of cytokine gene expression associated with fibrosis.

[0086] 1.7 FAD and nitric oxide concentration measurement

[0087] FAD levels in mouse serum and cells were determined using a FAD assay kit (#ab204710, Abcam), which was used for colorimetric detection according to the manufacturer's instructions. Protein concentrations were then standardized using a Micro BCA protein assay kit (ThermoScientific, 23235). NO levels in cell supernatants were determined using a NO assay kit (#A013-2-1, Nanjing Jiancheng Bioengineering Institute), which was used for colorimetric detection according to the manufacturer's instructions. Protein concentrations were then standardized using a Micro BCA protein assay kit (Thermo Scientific, 23235). Intracellular NO levels were quantitatively determined by flow cytometry using a DAF-FM DA fluorescent probe (Beyotime, S0019).

[0088] 1.8 iNOS, PDH, IDH and ACO2 activity measurement

[0089] Cellular iNOS activity was measured using an iNOS activity assay kit (#Ab211083, Abcam), while PDH (#BC0385, Solarbio), IDH (#BC2165, Solarbio), and ACO2 (#BC4480, Solarbio) activities were assessed using the corresponding activity assay kits. The assays were performed according to the manufacturer's protocol.

[0090] 1.9 Immunofluorescence staining

[0091] Liver tissues were embedded in OCT. The whole cryo-mold was frozen in a dry ice bath and prepared for cryosectioning. 5 μm thick cryosections were immunofluorescently stained with primary antibodies against F4 / 80 (#ab16911, Abcam), ARG1, and IL-1β. Sections were imaged using Alexa Fluor 488 or Alexa Fluor 594-conjugated secondary antibodies and fluorescence microscopy, and images were analyzed using ImageJ software.

[0092] 1.10 Mitochondrial and glycolysis stress test experiments

[0093] Peritoneal macrophages were seeded into 24-well Seahorse XFe-24 test plates (Agilent) and then subjected to the indicated stimulations, followed by washing with XF RPMI medium (pH 7.4) and incubation for 1 hour. The Seahorse XFe-24 software (Agilent) automatically calculates and records the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) tested by mitochondrial or glycolytic stress. To determine ATP production by OCR, 2.0 μM oligomycin (#HY-16589, MCE) was used. Maximum respiration rate was measured using 2.0 μM FCCP (#HY-100410, MCE). Next, cells were treated with 1.0 μM antimycin A (#MS0070-10MG, MKBio) / rotenone (#HY-B1756, MCE) to specifically measure non-mitochondrial respiration. Measurements were taken after the addition of each mitochondrial inhibitor and before the introduction of the next inhibitor. To assess ECAR, we used 10 μM glucose to determine the basic glycolytic potential. The maximum glycolytic capacity was measured by 1.5 μM oligomycin. Cells were treated with 50 μM 2-deoxy-D-glucose (2-DG) (#ST1024, Beyotime) to ensure that the ECAR obtained in the experiment was from the glycolytic pathway. Measurements were captured after adding each glycolytic inhibitor and before applying the next inhibitor.

[0094] 1.11 Mitochondrial respiratory complex (MRC) activity

[0095] Mitochondria were isolated from PM. Mitochondrial samples were quantified using the Micro BCA Protein Assay Kit (Thermo Scientific, 23235). The activity of MRC complex IV was assessed using a commercially available kit (Abbkine KTB 1850 / KTB 1860 / KTB1870 / KTB 1880 / KTB 1890) according to the manufacturer's instructions.

[0096] 1.12 Recombinant protein purification

[0097] Purified iNOS was produced by BL21 (DE3) competent E. coli cells transformed with pET28a-His-iNOS plasmid. The culture was grown at 37°C until the OD at 600nm reached 0.6, and then induced with 0.1mM IPTG at 18°C ​​for 18 hours. The resulting cell pellet was collected and sonicated in a lysis buffer containing 20mM Tris (pH7.5), 0.1% Triton X-100, 1mg / mL lysozyme and 1μg / mL DNase. The protein was then enriched using TALON metal affinity resin (Takara, 635503), eluted with 100mM imidazole, and finally concentrated into a buffer of 25mM Tris (pH 8.0), 0.4M NaCl. GST-RFK was produced by BL21 (DE3) competent E. coli cells transformed with pGEX-4T-1-GST-RFK and induced with 0.1 mM IPTG at 18°C ​​for 18 hours. The cell pellet was lysed as described above. Protein was enriched using GST-Sefinose resin (Sangon, C600031). The GST fusion protein was eluted with 10 mM reduced glutathione in 50 mM Tris (pH 8.0). The GST protein was purified from pGEX-4T-1-GST using the same procedure as GST-RFK. The eluted protein was dialyzed overnight in PBS and used for pull-down experiments.

[0098] 1.13GST / His pull-down assay

[0099] E. coli cell pellets transformed with pGEX-4T-1-GST-RFK or pET28a-His-iNOS were collected and lysed as described above. After centrifugation at 10,000 g for 15 minutes, the cells were incubated with GST-Sephinose resin for 4 hours, and the proteins were eluted and subjected to SDS-PAGE.

[0100] 1.14 Biotin-streptavidin pull-down assay

[0101] Biotin-RFK-peptide (aa1-30 / aa60-92 / aa101-130) or biotin-peptide-mix (SynbioTechnologies) were incubated with rotation for 2 h at 4°C and then incubated with purified iNOS or HEK293T lysate overexpressing HA-iNOS overnight at 4°C. The beads were then washed, immunoblotted, and the expression of iNOS was assessed.

[0102] 2. Experimental results

[0103] 2.1RFK promotes the polarization of M (IFN-γ) but inhibits the polarization of M (IL-4) through its enzymatic activity

[0104] To investigate the effect of RFK on macrophage polarization, a myeloid-specific Rfk-deficient cell line was constructed. mice and littermates Mice were used as controls. The results showed that the expression of M1-specific marker genes such as IL-1β, IL-6, Cxcl9, and TNF-α was inhibited in Rfk-deficient bone marrow-derived macrophages (BMDMs). Figure 2 In contrast, RFK deficiency upregulated the expression of M2-specific marker genes such as ARG1, Retnla, MGL1, and chemokine ligand 17 (CCL17) and chemokine ligand 22 (CCL22) ( Figure 2 DF in ).

[0105] Further studies showed that RFK overexpression significantly restored the downregulation of M1-specific marker gene expression caused by RFK knockdown. However, the levels of M1-specific marker genes in cells overexpressing RFK-E79Q (enzyme-inactive mutation) were similar to those in RFK-deficient cells, indicating that the enzymatic activity of RFK is required for its promotion of macrophage M1 polarization ( Figure 2 In addition, the enzymatic activity of RFK is also crucial for inhibiting the M2 polarization of macrophages ( Figure 2 IJ in Figure 1). These results suggest that RFK regulates macrophage polarization through its enzymatic activity. RFK inhibitors can promote macrophage M (IL-4) polarization and inhibit macrophage M (IFN-γ) polarization.

[0106] 2.2RFK deficiency alleviates liver fibrosis

[0107] An important function of M(IL-4) polarized macrophages is to alleviate the progression of fibrosis. The present invention further studied the regulatory effect of myeloid RFK on the M(IL-4) phenotype in the CCl4-induced liver fibrosis model and found that The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice were lower than Mouse ( Figure 3 In addition, myeloid RFK-deficient mice showed reduced liver damage and fibrosis ( Figure 3 BC in the liver). This suggests that RFK inhibitors can effectively inhibit AST and ALT levels, thereby improving liver damage and fibrosis. In addition, myeloid RFK deficiency significantly increased ARG1 + The proportion of macrophages ( Figure 3 D), which further suggests that riboflavin metabolism plays an important role in inhibiting the function of M(IL-4) macrophages in vivo. + 6G + Ly6Clow Neutrophils remain unchanged ( Figure 3 DE in ).

[0108] 2.3RFK regulates macrophage polarization through FAD

[0109] As a key enzyme in riboflavin metabolism, RFK is crucial in providing FAD. The present invention explored whether RFK can regulate macrophage polarization through FAD. Exogenous addition of FAD promoted M (IFN-γ) but inhibited M (IL-4) polarization. In addition, the addition of FAD restored the polarization phenotype of RFK-KO-BMDMs to the level of RFK-WT-BMDMs ( Figure 4 ) These results suggest that RFK regulates macrophage polarization through its downstream metabolite FAD.

[0110] 2.4RFK regulates macrophage polarization via NO

[0111] Under TNF-α stimulation, the interaction between RFK and FAD-dependent NADPH oxidase is enhanced, promoting the activity of NADPH oxidase and the generation of reactive oxygen species (ROS). Therefore, the present invention explores whether RFK regulates macrophage polarization through ROS and finds that although the antioxidants NAC and GSH inhibit the expression of M1-specific marker genes in macrophages, they can only slightly restore the differences caused by RFK overexpression ( Figure 5 A in Figure 2), suggesting that RFK regulates macrophage polarization through another mechanism. FMN and FAD are reported to be cofactors of iNOS, and RFK deficiency inhibits NO production. Whether stimulated by IFN-γ or LPS+IFN-γ, the level of NO in RFK-KO-BMDMs was lower than that in RFK-WT-BMDMs ( Figure 5 In the present invention, the addition of the NO donor diethylamine (DETA)NOate promoted the expression of M1-specific marker genes, but inhibited the expression of M2-specific marker genes, and restored the macrophage polarization phenotype caused by RFK deficiency ( Figure 5 In contrast, inhibition of NO production by treatment with the NO synthase inhibitor AG suppressed the expression of M1 and promoted the expression of M2-specific marker genes, and restored the macrophage polarization phenotype caused by RFK knockout or overexpression ( Figure 5 These results suggest that RFK regulates macrophage polarization through NO.

[0112] 2.5 RFK deficiency regulates energy metabolism through NO-mediated remodeling of the TCA cycle and mitochondrial electron transport chain

[0113] Energy metabolism is essential for macrophage polarization. Using the Seahorse extracellular flux analyzer to measure the extracellular acidification rate (ECAR) or oxygen consumption rate (OCR), we found that RFK deficiency significantly reduced the level of ECAR and increased the level of OCR in PMs treated with IFN-γ or IL-4 ( Figure 6 We further found that blocking mitochondrial ATP synthase activity with oligomycin (OM) and blocking glycolytic flux mediated by 2-deoxy-D-glucose (2-DG) abolished the energy metabolism reprogramming and macrophage polarization phenotype caused by RFK deficiency ( Figure 7 This suggests that RFK regulates macrophage polarization through glucose-driven energy metabolism. In addition, it was found that RFK deficiency increased the enzyme activities of PDH (pyruvate dehydrogenase), IDH2 (isocitrate dehydrogenase), and ACO2 (aconitase 2) ( Figure 6 EG), reducing the level of citric acid ( Figure 7 H in the mitochondrial electron transport complex ( Figure 6 in IJ).

[0114] It is reported that NO leads to mitochondrial dysfunction and glucose metabolism reprogramming by inhibiting the enzymatic activities of PDH, ACO2, IDH and mitochondrial electron transport complex. The present invention then explored whether NO plays a role in energy metabolism reprogramming caused by RFK deficiency. The present invention found that the addition of NO donor upregulated ECAR, inhibited the enzymatic activities of OCR and PDH, ACO2 and IDH, and restored the TCA cycle remodeling and changes in mitochondrial electron transport complex caused by RFK deficiency ( Figure 6 FAD regulates the protein stability of HIF-1α, which is essential for controlling immune cell metabolism and function. The present invention found that HIF-1α expression in NO donor-treated RFK-KO-BMDMs did not change significantly compared with RFK-WT-BMDMs ( Figure 6 These results suggest that RFK reprograms macrophage energy metabolism via NO.

[0115] 2.6RFK interacts with iNOS to enhance iNOS activity and reprogram macrophage energy metabolism and polarization phenotype

[0116] The riboflavin metabolites FMN and FAD are cofactors of iNOS. Using ClusPro protein docking analysis based on the crystal structure shown ( Fig. 9 A) We then explored whether RFK, a key enzyme in the riboflavin metabolic pathway, interacts with NOS to enhance iNOS activity and NO production. The present invention uses mass spectrometry analysis to identify RFK protein partners, including iNOS. ( Fig. 9 Immunoprecipitation assay confirmed that both ectopically expressed RFK and endogenous RFK interacted with NOS ( Figure 8 Purified recombinant RFK and iNOS proteins can also interact with each other ( Figure 8 The present invention also analyzed the interaction at various time points after cytokine treatment. IFN-γ stimulation increased the RFK-NOS interaction in both short-term (5-60 minutes) and long-term (6-12 hours) time periods ( Fig. 9 CD in ), while IL-4 did not significantly change the interaction at any time period ( Fig. 9 Then the present invention further explored which domain of iNOS interacts with RFK and found that the iNOS (aa 1-500) domain interacts with RFK ( Figure 8 E and Fig. 9 in GI).

[0117] Next, we used the ConSurf algorithm (https: / / consurf.tau.ac.il) to evaluate the amino acid conservation and exposure in the RFK structural model and synthesized biotin-labeled peptides RFK (aa 1-30), RFK (aa 60-92), and RFK (aa 101-130) to identify the key functional domains of RFK in the interaction with iNOS ( Fig. 9 Using a streptavidin pull-down assay, we found that RFK (aa 60-92) but not the other two peptides could bind to cellular iNOS ( Figure 8 F) and recombinant iNOS protein ( Figure 8 We attempted to identify the site of interaction at the amino acid level. Based on the conservation and exposure of the amino acids, we constructed RFK mutants, and immunoprecipitation experiments showed that mutations at S64 and Y91 affected the affinity of RFK to iNOS, but mutations at G66, P69, Y70, E79, or F86 did not affect the affinity of RFK to NOS ( Figure 8 H and Fig. 9 In addition, S64 A and Y91A mediated iNOS activity and NO levels much lower than those mediated by WT-RFK ( Figure 8 IJ and Fig. 9 These results suggest that S64 and Y91 of RFK are necessary for the binding of RFK to iNOS, thereby regulating the activity of RFK-bound iNOS.

[0118] Furthermore, overexpression of RFK restored the expression of M1 and M2-specific marker genes in RFK-deficient macrophages to WT levels, whereas its S64A and Y91A mutants only partially restored the phenotype, indicating that the S64 and Y91 sites are essential for RFK to regulate macrophage polarization. Figure 8 FAD supplementation restored the macrophage polarization phenotype changes caused by S64A and Y91A mutants, and this restoration was blocked by the NOS inhibitor AG and the glucose metabolism inhibitor 2-DG ( Figure 8 These results suggest that the S64 and Y91 sites of RFK are critical for the binding of RFK to iNOS, promoting its nitric oxide synthase activity and thereby regulating glucose metabolic reprogramming and macrophage polarization.

[0119] Comparative Example 1 Effect of phosphoglycerate dehydrogenase (PHGDH) on liver fibrosis

[0120] PHGDH can promote the polarization of M (IFN-γ) and inhibit the polarization of M (IL-4). Mice and Mice. The effect of myeloid PHGDH on serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice in a CCl4-induced liver fibrosis model was studied (refer to the 1.6 CCl4-induced liver fibrosis model method in Example 2).

[0121] The measurement results are shown in Table 1:

[0122] Table 1 Effect of PHGDH on AST and ALT (n=3)

[0123]

[0124]

[0125] The results showed that although PHGDH can promote macrophage M (IL-4) polarization and inhibit macrophage M (IFN-γ) polarization, the improvement effect of PHGDH deficiency on AST and ALT levels in CCl4-induced liver fibrosis mice was not as great as that of RFK deficiency.

[0126] Comparative Example 2 Effect of pyrophosphorylase (ADPG) on liver fibrosis

[0127] Pyrophosphorylase (ADPG) can hydrolyze flavin protein into riboflavin and is one of the key enzymes mediating riboflavin metabolism. Mice and Mice. The effects of myeloid ADPG on serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice in a CCl4-induced liver fibrosis model were studied (refer to the 1.6 CCl4-induced liver fibrosis model method in Example 2).

[0128] The measurement results are shown in Table 2:

[0129] Table 2 Effect of ADPG on AST and ALT (n=3)

[0130]

[0131] The results showed that ADPG deficiency had little effect on the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice in the CCl4-induced liver fibrosis model, proving that not all riboflavin metabolic enzymes can intervene in liver injury and fibrosis.

[0132] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. Use of riboflavin kinase inhibitors in the preparation of drugs for preventing and / or treating liver fibrosis.

2. The use according to claim 1, characterized in that: The riboflavin kinase inhibitor modulates macrophage polarization.

3. The use according to claim 2, characterized in that: The riboflavin kinase inhibitor reprograms energy metabolism and regulates macrophage polarization by inhibiting the catalytic activity of iNOS and the production of NO.

4. The use according to claim 3, characterized in that: The macrophage polarization inhibits the M1 type but promotes the polarization of the M2 type.

5. The use according to claim 1, characterized in that: The liver injury fibrosis is fibrosis caused by viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver injury or autoimmune liver disease.

6. The use according to any one of claims 1 to 5, characterized in that: The riboflavin kinase inhibitor is the sole or main active ingredient of the drug.

7. The use according to claim 6, characterized in that: The drug also includes pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable excipient is one or more of a filler, a binder, a disintegrant, a lubricant, a stabilizer, a surfactant, an antioxidant, a pH adjuster or a preservative.

9. The use according to claim 8, characterized in that: The pharmaceutically acceptable excipient is at least one of lactose, mannose, mannitol, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, mineral oil, vitamin C, vitamin E, citric acid, phosphoric acid, sodium benzoate, potassium sorbate, Tween-80, poloxamer, glutamic acid and glycine.

10. The use according to claim 9, characterized in that: The dosage form of the drug is injection, tablet, capsule, granule, powder, cream, ointment, gel or spray.

11. The use according to claim 10, characterized in that: The administration method of the drug is selected from oral, intravenous, local, intradermal or subcutaneous injection.

12. The use according to claim 11, characterized in that: The drug can also be used in combination with other drugs for preventing and / or treating liver injury and fibrosis.

13. The use according to claim 12, characterized in that: The other drugs for preventing and / or treating liver fibrosis are colchicine, silymarin, obeticholic acid, polyene phosphatidylcholine, entecavir, tenofovir, interferon, adefovir, lamivudine, Anluo Huaxian Pills, Fuzheng Huayu Capsules, Fufang Biejia Soft Liver Tablets, Xiaochaihu Decoction, Qiangan Capsules or Dahuang Zhechong Pills.

14. The use according to claim 12, characterized in that: The combined use is simultaneous use or sequential use.

15. The use according to claim 12, characterized in that: The dosage of the drug is adjusted according to the severity of the disease, the age of the patient, the method of administration and the course of treatment.

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