Patent Application
20220313793
The present invention relates to the field of biomedicine, and in particular relates to an application of flavin-containing monooxygenase 2 (FMO2) in preparing a drug for the treatment of non-alcoholic fatty liver disease (NAFLD).
Non-alcoholic fatty liver disease (NAFLD) refers to a clinical syndrome having a main pathological feature of excessive deposition of lipids (mainly triglycerides) in liver parenchymal cells not caused by drinking or other clear reasons. NAFLD is a progressive liver disease, divided into four stages from mild to severe: non-alcoholic fatty degeneration of liver, nonalcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma. According to related surveys, the prevalence of NAFLD worldwide is 25%, and about 30% of the population in China suffers from the disease. In particular, with the progress of society and the continuous improvement of living standards, intake of a lot of high-fat foods and imbalance of the dietary have caused the incidence of NAFLD to increase year by year and a rapid trend of youth of the affected population.
NAFLD is not a single cause disease, and its pathogenesis is complicated. The typical “two hits” theory believes that a large amount of fat accumulation in the liver parenchymal cells caused by various reasons (especially an increase in the insulin concentration) is the first hit, and lipid peroxidation and oxidative stress are the second hit. In recent years, with further research, a “multiple hits” theory has gradually been accepted by people, and it believes that the pathogenesis of NAFLD is not limited to the two hits mentioned above, and shall also include other factors such as inflammatory cytokines, host-microbe interactions, genetic factors, and dietary factors. At present, there are no really effective therapeutic drugs for NAFLD in clinical practice, and the treatment mainly focuses on removal of the inducement and adjustment of the diet. Therefore, it is urgent to develop high-efficiency and low-side-effect drugs for the treatment of NAFLD.
Flavin-containing monooxygenase (FMO) belongs to the family of flavoproteinases, and is a group of microsomal enzymes relying on flavin adenine dinucleotide (FAD), reduced nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen. FMO is generally found in eukaryotes. Due to the characteristics of regional selection, chiral catalysis, good stability, and the like, FMO has received extensive attention in the fields of xenobiotic metabolism, pharmacokinetics, biocatalytic synthesis, and the like. FMO is the second important drug metabolism enzyme after the cytochrome P450 (CYPs) enzyme, and FMO is a very important enzyme in detoxification and elimination of drugs and chemical substances, and can catalyze oxidation of compounds and drugs containing nitrogen, sulfur, phosphorus, selenium and other nucleophilic heteroatoms. Subtypes of FMO exist in many mammals and humans, and each has its substrate specificity and tissue localization. Although all isomers of human FMO have about 60% sequence similarity, their structural and functional similarities are very low. Studies have reported that the lack or hypofunction of FMO3 can lead to trimethylamine metabolism disorders, which is the cause of “fish smell syndrome”, that is, trimethylaminuria.
Furthermore, FMO2 is a member of the flavin-containing monooxygenase family, and is an enzyme with NADP+ and FAD as prosthetic groups and having biological metabolism. After the protein thereof was discovered in 1992, there was no follow-up study of its function for a long time. Until 2015, studies have found that FMO2 is related to the lifespan and activity of nematodes. So far, the research on FMO2 has mainly focused on gene polymorphism, and there are few studies on the function of FMO2. Compared with CYPs, FMOs have long been ignored. However, with research on the characteristics of FMO family members and the consequences of rare mutations and common polymorphic mutations of FMO, people have become more and more aware of the pharmacological and toxicological significance of these enzymes. As being an important enzyme in the liver, FMO has high research value.
In order to solve the above technical problems, the present invention provides an application of flavin-containing monooxygenase 2 (FMO2) in preparing a drug for treatment of non-alcoholic fatty liver disease (NAFLD). The present invention finds for the first time that FMO2 has an effect of preventing or treating NAFLD.
The specific technical solutions of the present invention are as follows:
In the first aspect, the present invention provides an application of FMO2 in preparing a drug for treatment of NAFLD.
The present invention finds that FMO2 can achieve the effect of treating fatty liver by inhibiting de novo lipid synthesis mediated by SREBP1/ACC/FASN pathways in the liver.
Preferably, the drug is a drug that reduces liver lipid synthesis and storage or a drug that promotes liver lipid metabolism.
Preferably, the FMO2 has a nucleotide sequence as shown in SEQ ID NO. 1 (mice) or SEQ ID NO. 3 (human), and an amino acid sequence as shown in SEQ ID NO. 2 (mice) or SEQ ID NO. 4 (human).
In the second aspect, the present invention provides a drug for the treatment of NAFLD, which contains FMO2.
Preferably, the drug is a drug that reduces liver lipid synthesis and storage or a drug that promotes liver lipid metabolism.
Preferably, the FMO2 has a nucleotide sequence as shown in SEQ ID NO. 1 (mice) or SEQ ID NO. 3 (human), and an amino acid sequence as shown in SEQ ID NO. 2 (mice) or SEQ ID NO. 4 (human).
Preferably, the drug is an oral preparation or an injection preparation.
Preferably, the dosage form of the oral preparation is a capsule, a tablet, a solution or powder; and the injection preparation is an injection or powder.
Preferably, the drug further includes a pharmaceutically acceptable carrier, excipient or solvent.
Compared with the prior art, the present invention has the beneficial effect that the present invention finds that FMO2 has an effect of preventing or treating NAFLD.
The present invention will be further described below in conjunction with embodiments.
A drug for treatment of non-alcoholic fatty liver disease (NAFLD), contains flavin-containing monooxygenase 2 (FMO2).
Preferably, the drug is a drug that reduces liver lipid synthesis and storage or a drug that promotes liver lipid metabolism.
Preferably, the FMO2 has a nucleotide sequence as shown in SEQ ID NO. 1 (mice) or SEQ ID NO. 3 (human), and an amino acid sequence as shown in SEQ ID NO. 2 (mice) or SEQ ID NO. 4 (human).
Preferably, the drug is an oral preparation or an injection preparation. Further, the dosage form of the oral preparation is a capsule, a tablet, a solution or powder; and the injection preparation is an injection or powder.
Preferably, the drug further includes a pharmaceutically acceptable carrier, excipient or solvent.
Preparation of Flavin-Containing Monooxygenase 2 (FMO2) Overexpression Virus:
In the embodiments of the present invention, FMO2 overexpression lentivirus (Ubi-FMO2-3FLAG-CBh-gcGFP-IRES-puromycin), and negative control lentivirus (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin) were all purchased from Shanghai Genechem Co., LTD.; and a mouse FMO2 nucleotide sequence was as shown in SEQ ID NO. 1.
1. Establishment of a Mouse Model of NAFLD
In the present invention, 8 weeks-old healthy C57BL/6J mice were divided into four groups, that is, a wild-type mouse normal control diet group, a wild-type mouse high-fat diet group, an FMO2 knockout mouse normal control diet group, and an FMO2 knockout mouse high-fat diet group. 5 mice in each group were fed for 12 weeks.
2. Extraction, Fixation, Sectioning, and Staining of Tissue
(1) Extraction: After the mice were anesthetized with 1% pentobarbital sodium, the abdominal cavity was quickly opened, and the liver was quickly separated after blood was collected from the heart. After weighing, an appropriate amount of liver tissue was cut and put into prepared tissue lysis buffer, triglyceride lysis buffer and 4% paraformaldehyde fixative.
(2) Fixation and sectioning: After being fixed with 4% paraformaldehyde, the liver was divided into two parts for paraffin embedding and OCT embedding respectively. The paraffin-embedded tissue was sectioned serially (with a thickness of 3 μm). After being picked out, the sections were placed in a 60° C. oven, baked for 30 minutes, and placed in a clean section box for later use. During OCT embedding, the tissue was slowly immersed in liquid nitrogen to prevent the tissue block from freezing and cracking. 6 μm thick frozen sections were attached to a cationic glass slide, and dried and stored in a −80° C. refrigerator for later use.
(3) HE staining: After being fixed with 4% paraformaldehyde for 10 minutes, the liver paraffin sections were washed with PBS for 5 minutes 3 times, subjected to nucleus staining with hematoxylin for 40 seconds, subjected to 1% hydrochloric acid (1 mL of concentrated hydrochloric acid with 99 mL of absolute ethanol added) alcohol differentiation and immersed 3 times, washed with water for 5 minutes 3 times, stained with eosin for 1 minute, washed with water for 5 minutes 3 times, dehydrated with gradient alcohol (75%, 2 seconds; 85%, 2 seconds; 90%, 2 minutes; 95%, 2 minutes; 100%, 5 minutes/2 times), washed with xylene for 5 minutes 2 times, and mounted with resin.
3. Liver Triglyceride Detection
About 50 mg of liver tissue was taken, 1 ml of triglyceride lysis buffer was added, and the liver tissue was homogenized 30 times in a glass homogenizer and lysed for 10 minutes at room temperature. 100 μl of the homogenate was taken for BCA protein measurement, and the remaining homogenate was placed in a 70° C. water bath for 10 minutes to inactivate lipase. The homogenate was centrifuged at 2000 rpm for 5 minutes at room temperature, the supernatant was taken for triglyceride quantification, and finally the quantity was converted to TG/protein in μmol/g.
4. RNA Extraction, Reverse Transcription and Real-Time Fluorescent Quantitative PCR
1 ml of Trizol (Invitrogen) was added to 20 mg of tissue. After the tissue was homogenized, 200 μl of trichloromethane was added, shaken vigorously for mixing, and centrifuged at 12000 rpm for 15 minutes. The supernatant was transferred to the same volume of isopropanol, mixed by inversion, and centrifuged at 12000 rpm to obtain a precipitate. The precipitate was washed twice with 70% ethanol, and 50-100 μl of DEPC water was added. After the RNA concentration was measured, 1500 ng of total RNA was taken for reverse transcription. The reverse transcription uses the PrimeScript™ RT Master Mix reverse transcription kit from Takara. Fluorescent quantitative PCR uses the TB Green® Premix Ex Taq™ II kit from Takara. The instrument used in the experiment is the ABI7500 fluorescent quantitative PCR instrument. The real-time fluorescent quantitative PCR primer sequences used in the experiment are as follows:
5. Detection of Expression of SREBP1/ACC/FASN in Liver Tissue by Western Blot
About 50 mg of liver tissue was taken, 1 ml of Ripa protein lysis buffer (containing a protease inhibitor) was added, and the liver tissue was ground thoroughly and centrifuged at 12000 g and 4° C. for 20 minutes. The supernatant was taken to detect the protein concentration by a BCA method. The protein was quantified to 2 mg/ml with a Ripa tissue protein lysis buffer and a 5×SDS protein loading buffer, and boiled at 98° C. for 5 minutes in a metal bath. 20 μg of protein solution was taken in an 8% polyacrylamide gel for electrophoresis, and transferred to a PVDF membrane. The primary antibodies are FMO2 (NOVUS, NBP1-85952), SREBP1 (Abcam, ab28481), ACC (CST, 3676), and FASN (Abcam, ab99359) antibodies, and the secondary antibody is goat anti-rabbit antibody (CST, 7074). After washing thoroughly with PBST, exposure was carried out in a dark room according to instructions of super ECL plus.
6. Isolation of Primary Hepatocytes and Establishment of In Vitro Models
(1) Isolation of Primary Hepatocytes
(1.1) The required instruments were autoclaved in advance, and DHANKS and 0.03% collagenase IV were prepared. A catheter was soaked with 75% alcohol; a super-clean bench was disinfected by UV irradiation for 30 min; and a medium, DHANKS and collagenase IV were preheated at 37° C.
(1.2) Sodium pentobarbital (60 mg/kg) was injected intraperitoneally to anesthetize the mice.
(1.3) The soaked and disinfected catheter was filled with the preheated DHANKS.
(1.4) The skin was prepared and disinfected with iodine. The abdominal cavity was opened in the super-clean bench to fully expose the portal vein, 1 cm of the portal vein was separated and threaded, and the portal vein was fixed by intubation.
(1.5) The portal vein was perfused with the DHANKS at a flow rate of 2 mL/min, and the liver became white after perfusion with 20 mL of DHANKS. Then the portal vein was perfused with the preheated collagenase IV at a flow rate of 2 mL/min, and the liver was removed after perfusion with 20 mL of preheated collagenase IV.
(1.6) The liver was washed with preheated DHANKS, and placed in a mixture of 10 mL of collagenase IV and 10 mL of DHANKS, and the envelope was gently torn off with curved forceps to suspend the hepatocytes in the mixture.
(1.7) The hepatocyte suspension was digested in a 37° C. water bath with shaking for 20 minutes, and filtered with a 200-mesh screen.
(1.8) The filtered hepatocyte suspension was centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The hepatocytes were suspended in a serum-free DMEM medium and centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded.
(1.9) The obtained hepatocyte pellet was resuspended in a high-sugar DMEM medium containing 10% FBS, and the viability was observed by trypan blue staining. The proportion of living cells was calculated, and the suspended cells were spread in a 6-well plate with 106 cells per well.
(1.10) The cells were incubated at 37° C. After 1 hour, the medium was changed to 10% FBS high-sugar DMEM. After another 3 hours, the medium was changed to a high-sugar DMEM medium containing 2% FBS. The cell morphology was observed after 12 hours.
(2) Cell counting method: The diluted cell suspension was dropped on a counting plate to make the suspension freely fill the gap under a cover glass without leaving bubbles. Then, the cells are observed under a light microscope and the number of cells in large squares at the four corners was counted. When the cells are disposed on a line, only the cells on an upper line and a right line are counted, and then the cell concentration is calculated by the following formula: (total number of cells in the large squares/4)×10000×dilution factor=number of cells/mL
(3) Cell modeling: 500 μM of oleic acid was added to the isolated primary hepatocytes after 24 h. Then, the primary hepatocytes were cultured for another 24 hours and stained with oil red O. Samples were collected to extract RNA, protein and triglycerides.
(4) FMO2 Overexpression Virus Transfection
Negative control lentivirus (FMO2-Vector) and FMO2 overexpression lentivirus (FMO2-Over) were added to the isolated primary hepatocytes after 24 hours, with an MOI of 20, and the medium was changed at the time of 12 hours after transfection. After 48 hours, 500 μM of oleic acid was added to make a model.
7. Oil Red O Staining of Cells
(1) Preparation of a working solution: A stock solution of saturated oil red O was added to double distilled water with a ratio of 3:2 (oil red O: double distilled water), mixed well, placed at room temperature for 5 minutes, and filtered for use.
(2) After the medium was pipetted, the cells were rinsed with PBS 3 times, fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed with PBS for 5 minutes, stained with the oil red O working solution for 15 minutes, washed with PBS for 5 minutes 3 times, and photographed under an inverted optical microscope within 1 hour.
8. Detection of Triglyceride in Primary Hepatocytes
The cells were washed with PBS 3 times, and 200 μl of a lipid lysis buffer was added to each well of the six-well plate. The cells were scraped off with a scraper and homogenized by ultrasound, and lysed at room temperature for 10 minutes. 50 μl of homogenate was taken for BCA protein measurement, and the remaining homogenate was placed in a 70° C. water bath for 10 minutes to inactivate lipase. The homogenate was centrifuged at 2000 rpm for 5 minutes at room temperature, the supernatant was taken for triglyceride quantification, and finally the quantity was converted to TG/protein in μmol/g.
9. Detection of Expression of SREBP1/ACC/FASN in Primary Hepatocytes by Western Blot
The cells were washed with PBS 3 times, and 100 μl of a Ripa protein lysis buffer (containing a protease inhibitor) was added to each well of the six-well plate. The cells were scraped off with a scraper, homogenized by ultrasound, ground thoroughly and centrifuged at 12000 g and 4° C. for 20 minutes. The supernatant was taken to detect the protein concentration by a BCA method. The protein was quantified to 1 mg/ml with a Ripa tissue protein lysis buffer and a 5×SDS protein loading buffer, and boiled at 98° C. for 5 minutes in a metal bath. 20 μg of protein solution was taken in a 8% polyacrylamide gel for electrophoresis, and transferred to a PVDF membrane. The primary antibodies are FMO2 (NOVUS, NBP1-85952), SREBP1 (Abcam, ab28481), ACC (CST, 3676), and FASN (Abcam, ab99359) antibodies, and the secondary antibody is goat anti-rabbit antibody (CST, 7074). After washing thoroughly with PBST, exposure was carried out in a dark room according to instructions of super ECL plus.
10. De Novo Lipid Synthesis Experiment
Primary hepatocytes were isolated and inoculated in a 6-well plate at a density of 106 cells per well, and cultured overnight in serum-free high-sugar DMEM after adherence. On the next day, the medium was changed to a high-sugar medium containing 0.2% fatty acid-free BSA. After 2 hours, the medium was changed to a DMEM medium containing 0.5 mM OA, and 0.5 uCi U-14C-labeled glucose was added to each well. After culturing for 4 hours at 37° C., the lipids were extracted by a Folch method: The cells were washed twice with pre-cooled PBS, and the cells in each well were scraped with 200 μl of PBS containing 0.5% Triton. 150 μl of cells were taken and 500 μl of a solution of chloroform and methanol in a ratio of 2:1 was added to vigorously shake for 30 seconds, and then 125 μl of double distilled water was added after standing on ice for 1 hour. After vigorous shaking, the mixture was centrifuged at 4° C. and 1000 rpm for 15 minutes, the lower organic phase was taken, and the entry of carbon 14 into the lipids was measured using a Tri-Carb 2900 TR liquid scintillation counter. All readings were normalized with protein concentration.
11. Data Analysis
1. Deletion of FMO2 Gene Aggravates NAFLD Induced by High-Fat Diet
In order to study the role of the FMO2 gene in non-alcoholic liver disease, 8-weeks-old FMO2 knockout mice (FMO2−/−) and their littermate wild-type control mice (FMO2−/−) were fed with a normal control diet (NCD) and a high-fat diet (HFD) for 12 weeks. After that, the mice were sacrificed and the accumulation of lipids in the liver of the mice was observed. In this study, FMO2 knockout mice were constructed by homologous recombination. Western Blot results in the liver showed that FMO2 was completely knocked out (
2. Deletion of FMO2 Gene Aggravates Hepatocyte Steatosis Induced by Oleic Acid
In order to study the effect of deletion of the FMO2 gene on hepatocyte steatosis in vitro, primary hepatocytes were isolated from the livers of the wild-type mice and FMO2 mice. Western Blot results showed that the isolated FMO2−/−primary hepatocytes lacked FMO2 gene expression (
3. Overexpression of FMO2 Gene Improves Hepatocyte Steatosis Induced by Oleic Acid
From the in vivo mouse high-fat feeding model and the in vitro hepatocyte oleic acid induced model, it can be seen that the deletion of the FMO2 gene can aggravate fatty liver. In order to verify the above conclusion on the negative side, primary hepatocytes were isolated from the livers of wild-type mice and infected with the Ubi-FMO2-3FLAG-CBh-gcGFP-IRES-puromycin lentivirus to overexpress the FMO2 gene. At the same time, the hepatocytes of the control group were infected with the Ubi-MCS-3FLAG-CBhg-cGFP-IRES-puromycin negative control lentivirus. Western Blot results showed that infection with the FMO2 overexpression lentivirus could effectively increase the protein level of FMO2 in hepatocytes (
4. FMO2 Improves NAFLD by Inhibiting De Novo Lipid Synthesis Mediated by SREBP1/ACC/FASN Pathways
NAFLD is mainly characterized by excessive fat deposition in hepatocytes, inflammation and abnormal liver function, and its pathogenesis involves many factors such as heredity, metabolism, environment, and lifestyle. At present, the exact pathogenesis of NAFLD has not yet been fully unequivocal. As a metabolic center of animals, lipid metabolism in the liver is an extremely complex process, including lipid uptake, lipid de novo synthesis, lipid oxidation, etc. Any abnormality in any link may lead to abnormal lipid deposition in the liver.
In order to explore the specific mechanism of FMO2 gene deletion aggravating NAFLD and find out which link in liver lipid metabolism is regulated by FMO2, the expression of a series of lipid metabolism-related genes in the livers of mice fed with the high-fat diet were tested. PCR results showed that compared with the wild-type mice, there was no significant difference in cholesterol synthesis, lipid uptake, and lipid oxidation in the livers of the FMO2−/−mice, but the expression of genes related to lipid de novo synthesis increased (
The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the field; and the methods used in the present invention, unless otherwise specified, are all conventional methods in the field.
The above are only preferred embodiments of the present invention, and do not impose any limitation on the present invention. Any simple modifications, alterations and equivalent transformations made to the above embodiments according to the technical essence of the present invention still belong to the protection scope of the technical solutions of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010825464.1 | Aug 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/083861 | 3/30/2021 | WO |
