Application of fucoxanthine in preparation of medicine for treating and preventing autoimmune hepatitis

Abstract

The invention relates to application of fucoxanthine in preparation of a medicine for treating autoimmune hepatitis, the fucoxanthine serves as a natural carotenoid and has a remarkable liver protection effect, and the research reveals that the fucoxanthine reduces liver injury and ferroptosis in AIH by activating a mitochondrial autophagy signal channel to inhibit ferroptosis for the first time. Experimental results show that the fucoxanthine effectively inhibits ferroptosis by reducing lipid peroxidation, up-regulating expression of glutathione peroxidase 4 (GPX4), down-regulating expression of cyclooxygenase 2 (COX-2) and improving mitochondrial morphological change related to ferroptosis. The invention provides a new natural medicine choice for treating AIH, and provides powerful support for clinical treatment and prevention of autoimmune hepatitis.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the use of fucoxanthin in preparing a medicine for treating autoimmune hepatitis. Background Art

[0002] Autoimmune hepatitis (AIH) is a chronic, progressive liver disease that can lead to liver fibrosis, cirrhosis, and liver failure. As a heterogeneous disease, AIH manifests differently in different patients, making diagnosis and treatment a challenge. Although current treatments for AIH are mostly based on immunosuppressants, some patients do not respond well to treatment, and long-term use of immunosuppressants can lead to serious side effects. Therefore, it is of great significance to find safer and more effective treatments.

[0003] In 2012, Dr. Brent R. Stockwell of Columbia University proposed that ferroptosis is a unique form of programmed cell death, different from apoptosis, necroptosis and necrosis, and is characterized by a cell death pattern caused by lipid peroxidation and the accumulation of reactive oxygen free radicals (ROS). During ferroptosis, mitochondrial cristae are reduced or absent, and even mitochondria are shrunk. Ferroptosis is associated with a variety of diseases, including cancer, cardiovascular disease, and immune system diseases, and it is increasingly recognized that it is related to liver disease. Many studies have shown that ferroptosis exists in AIH, but its specific molecular mechanism remains unclear. Inhibiting ferroptosis of hepatocytes has also become a novel and promising treatment for AIH.

[0004] Fucoxanthin (FX) is a marine carotenoid found mainly in brown seaweed, accounting for about 10% of the total natural carotenoid production. FX has multiple biological activities, including antioxidant, anti-inflammatory and hepatoprotective effects. Studies have shown that FX is particularly effective in the treatment of oxidative stress-related diseases. Zhang et al. reported that mitophagy was activated in endothelial cells after traumatic brain injury. FX alleviated blood-brain barrier damage, endothelial cell apoptosis and ferroptosis by regulating mitophagy. Li et al. found that FX can alleviate free fatty acid-induced macrophage inflammation by improving mitochondrial function and regulating lipid metabolism. However, the mechanism of action of FX in AIH, especially its regulatory mechanism of ferroptosis, is still unclear. Summary of the invention

[0005] Purpose of the present invention: In order to achieve the improvement of ferroptosis to treat autoimmune hepatitis, the present invention provides a use of fucoxanthin in the preparation of drugs for the treatment and prevention of autoimmune hepatitis, which can promote the inhibition of ferroptosis to improve autoimmune hepatitis.

[0006] The invention discloses use of fucoxanthin in preparing a medicine for treating and preventing autoimmune hepatitis.

[0007] Furthermore, the fucoxanthin reduces liver damage and plays a liver-protective role by inhibiting ferroptosis and activating mitochondrial autophagy signaling pathways.

[0008] Furthermore, the fucoxanthin can inhibit ferroptosis by reducing the generation of lipid peroxides, upregulating the expression of glutathione peroxidase 4 (GPX4), downregulating the expression of cyclooxygenase 2 (COX-2), and alleviating the mitochondrial morphological changes associated with ferroptosis.

[0009] Furthermore, the fucoxanthin can maintain the integrity of mitochondria by enhancing mitochondrial autophagy.

[0010] Furthermore, the fucoxanthin inhibits ferroptosis by alleviating lipid peroxidation by regulating mitochondrial autophagy.

[0011] Furthermore, the fucoxanthin is used as an active ingredient to prepare a pharmaceutical composition for activating mitochondrial autophagy and inhibiting ferroptosis.

[0012] Furthermore, the drug is an oral preparation or an injectable preparation.

[0013] Furthermore, the oral preparation is any one of tablets, capsules or granules.

[0014] In summary, the beneficial effects of the present invention are as follows: S100 induces ferroptosis of liver tissue cells, and according to data analysis using GSH detection, MDA detection, qRT-PCR, WB detection and electron microscopy observation, the fucoxanthin reduces lipid peroxidation by regulating mitochondrial autophagy to inhibit ferroptosis in autoimmune hepatitis induced by S100. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph showing the experimental results of the fucoxanthin of the present invention improving liver damage and ferroptosis in S100-induced autoimmune hepatitis.

[0016] Figure 2 This is a graph showing the experimental results of FX of the present invention inhibiting mitochondrial autophagy in S100-induced autoimmune hepatitis.

[0017] Figure 3 This is a diagram showing the experimental results that FX of the present invention inhibits ferroptosis in liver tissue cells of autoimmune hepatitis by promoting mitochondrial autophagy. DETAILED DESCRIPTION

[0018] The specific embodiments of the present invention will be described in detail below. It should be noted that the following embodiments are only used to help understand the content of the present invention and do not limit the present invention. In addition, the technical features in the various embodiments can be combined as long as there is no conflict between them.

[0019] 1. Experimental Preparation

[0020] 1.1 Experimental animals

[0021] 57BL / 6J mice (male, 6-8 weeks, 20 ± 2 g) were housed in ventilated cages in the specific pathogen-free facility of the First Affiliated Hospital of Wenzhou Medical University. The animals were housed in a temperature- and humidity-controlled room and were allowed ad libitum access to food and water.

[0022] All animal experimental procedures were approved and supervised by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Wenzhou Medical University, and all animal experiments were performed in accordance with relevant guidelines.

[0023] 1.2. Experimental instruments

[0024] Dehydration machine, Hubei Benoit Medical Technology Co., Ltd.;

[0025] Paraffin embedding machine (cold stage), SLEE, Germany;

[0026] Biological tissue automatic embedding machine, SLEE, Germany;

[0027] Rotary microtome, Leica Microsystems Shanghai Co., Ltd.;

[0028] KD-P tissue slide machine, Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.;

[0029] Oven, Shanghai Yuejin Medical Instrument Co., Ltd.;

[0030] Dakowe dyeing machine, Shenzhen Dakowe Medical Technology Co., Ltd.;

[0031] Glass slides and cover slips were provided by Jiangsu Huida Medical Instrument Co., Ltd.

[0032] Microscope, NIKON, Japan;

[0033] Electric constant temperature water bath, Shanghai Yuejin Instrument Co., Ltd.;

[0034] Decolorization shaker, Shanghai QiTe Analytical Instrument Co., Ltd.;

[0035] Microwave ovens, Midea Group, China;

[0036] Micropipette, Eppendorf, Germany;

[0037] Vortex shaker, Thermo Forma, USA

[0038] Normal temperature centrifuge, Thermo Forma, USA;

[0039] High temperature and high pressure sterilizer, Systec, Germany;

[0040] Tissue homogenizer, Ningbo Xinzhi Biotechnology Co., Ltd.;

[0041] Electronic balance, Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0042] Ice machine, Scotsman, Italy;

[0043] -80℃ ultra-low temperature refrigerator, Thermo Fisher, USA;

[0044] Electrophoresis apparatus, Bio-rad, USA;

[0045] Transfer membrane instrument, Bio-rad, USA;

[0046] Shaker, Chilin Bell Laboratory Instrument Company;

[0047] Gel imaging system, Shanghai Tianneng Life Science Co., Ltd.;

[0048] Ultrapure water purifier, Shanghai Hetai Experimental Instrument Co., Ltd.;

[0049] Ice machine, Scotsman, Italy;

[0050] PCR amplifier, Biorad, USA;

[0051] Real-time fluorescence quantitative PCR system, Thermo Fisher, USA; fully automatic biochemical analyzer, Beckman Coulter, USA;

[0052] Constant temperature drying oven, Shanghai Yuejin Instrument Co., Ltd.;

[0053] Centrifuge tubes of various specifications, Nest Company, USA;

[0054] Various EP tubes and gun tips of various specifications, Axygen, USA;

[0055] Flasks, beakers, measuring cylinders, Beijing Tiangen Biochemical Co., Ltd.

[0056] 1.3 Experimental Reagents

[0057] Fucoxanthin, MCE, USA;

[0058] β-cyclodextrin, MCE, USA;

[0059] Complete Freund's adjuvant, Sigma, USA;

[0060] Autophagy inhibitor 3-MA, Selleck Biotechnology Co., Ltd., USA; physiological saline, Shanghai Ruji Biotechnology Co., Ltd.; 4% paraformaldehyde, Beijing Solarbio Technology Co., Ltd.; PBS phosphate buffer, ThermoFisher, USA;

[0061] PBS phosphate powder, Beijing Solarbio Technology Co., Ltd.;

[0062] Eosin staining, Beijing Solarbio Technology Co., Ltd.;

[0063] Neutral gum mounting medium, Beijing Solarbio Technology Co., Ltd.;

[0064] Anhydrous ethanol, Beijing Solarbio Technology Co., Ltd.;

[0065] Xylene reagent, Beijing Solarbio Technology Co., Ltd.;

[0066] Hematoxylin stain, Hangzhou Hawke Biotechnology Co., Ltd.;

[0067] Ammonia, Shanghai MacLean Biochemical Technology Co., Ltd.;

[0068] Methanol, Wenzhou Xiang'an Chemical Co., Ltd.;

[0069] Citrate buffer (100X), Beijing Biolab Technology Co., Ltd.;

[0070] Cell-grade DMSO, Beijing Solarbio Technology Co., Ltd.;

[0071] GSH detection kit, Nanjing Jiancheng Bioengineering Institute;

[0072] MDA detection kit, Nanjing Jiancheng Bioengineering Institute;

[0073] Mammalian protein extraction reagents, Wuhan Boster Biotechnology Co., Ltd.;

[0074] BCA protein concentration determination kit, Shanghai Bio-Tech Biotechnology Co., Ltd.; protease and phosphatase inhibitor mixture, Suzhou New Saimei Biotechnology Co., Ltd.; protein marker, Thermo Fisher, USA;

[0075] 5x dual-color loading, Hangzhou Fude Biotechnology Co., Ltd.;

[0076] PAGE gel rapid preparation kit (10%), Shanghai Yazyme Biopharmaceutical Co., Ltd.; glycine, Beijing Solarbio Technology Co., Ltd.;

[0077] Tris-HCl, Beijing Solarbio Technology Co., Ltd.;

[0078] SDS, Beijing Solarbio Technology Co., Ltd.;

[0079] TBST (20×) (containing 1% Tween 20), Beijing Solarbio Technology Co., Ltd.; PVDF membrane, Millipore, Merck Group, Germany;

[0080] Skim milk powder, BD Company, USA;

[0081] ECL chemical developer, Thermo Fisher, USA;

[0082] PCR primers, Sangon Biotech (Shanghai) Co., Ltd.;

[0083] RNAiso Plus, Takra, Japan;

[0084] Bromo-3-chloropropane, Shanghai MacLean Biochemical Technology Co., Ltd.;

[0085] Isopropyl alcohol, Sinopharm Chemical Reagent Co., Ltd.;

[0086] Reverse transcription kit, Takra, Japan;

[0087] DEPC water, Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0088] Anti-GPX4 antibody, Abcam, UK;

[0089] Anti-COX-2 antibody, Abcam, UK;

[0090] Anti-GAPDH antibody, Proteintech, USA;

[0091] Anti-pink1 antibody, Hangzhou Huaan Biotechnology Co., Ltd.;

[0092] Anti-parkin antibody, Hangzhou Huaan Biotechnology Co., Ltd.;

[0093] Anti-LC3 antibody, Proteintech, USA;

[0094] Anti-p62 antibody, Hangzhou Huaan Biotechnology Co., Ltd.;

[0095] Western primary antibody diluent, Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0096] Goat anti-rabbit secondary antibody, Shanghai Biotime Biotechnology Co., Ltd.;

[0097] 1.4 Preparation of S100

[0098] (1) A male B6 mouse was randomly selected for extraction of S100 liver antigen;

[0099] (2) The mice were intraperitoneally injected with 10% chloral hydrate at a rate of 10 ml / kg, and after anesthesia, the mice were fixed on an operating platform;

[0100] (3) After the cross-shaped laparotomy, the abdominal and thoracic cavities are clearly exposed, and the liver is perfused through the heart until the blood color of the liver disappears and the perfusion turns white;

[0101] (4) After removing the gallbladder, the liver was removed and placed in pre-cooled PBS for later use. The mouse liver was evenly divided into 10 EP tubes filled with PBS, and then the liver tissue was ground into a tissue homogenate using an electric tissue homogenizer;

[0102] (5) Transfer the tissue homogenate to a 50 ml centrifuge tube and centrifuge at 150 g for 10 min at 4°C to remove cell nuclei;

[0103] (6) Place the supernatant from the centrifugation in an ultracentrifuge at 100,000 g for 1 h at 4°C;

[0104] (7) The supernatant obtained after ultracentrifugation is placed in a 15 ml centrifuge tube, i.e., S100;

[0105] (8) Measure the protein concentration using the BCA method, add PBS to make the concentration of 1.5 mg / mL that we need to inject into mice, prepare a filter, pass the protein through the filter, and then add an equal volume of Freund's adjuvant to fully grind and emulsify to prepare S100 mixed solution;

[0106] (9) On day 0 and day 7 of the experiment, 1 ml of the mixture was injected intraperitoneally into mice.

[0107] 1.5 Preparation of fucoxanthin for intragastric administration

[0108] (1) Dissolve 12.5 mg of fucoxanthin powder in 1 mL of DMSO solution and dissolve with ultrasound to prepare a fucoxanthin stock solution;

[0109] (2) Dissolve 2 g of β-cyclodextrin in 10 mL of normal saline and sonicate at 40 Hz for 1 min to obtain 20% SBE-β-CD normal saline;

[0110] (3) 100 uL of the fucoxanthin stock solution was mixed evenly with 900 uL of 20% SBE-β-CD saline to prepare a 1.25 mg / mL working solution for intragastric administration to mice.

[0111] 1.6 Preparation of 3-MA solution for intraperitoneal injection

[0112] 4 mg of 3-MA powder was dissolved in 1 mL of sterile PBS and ultrasonically assisted to prepare a 3-MA working solution with a concentration of 4 mg / mL for intraperitoneal injection in mice.

[0113] 2. Construction of Mouse Experiment

[0114] 2.1 Experimental Grouping

[0115] C57BL / 6J mice (male, 6-8 weeks old, 20±2g) were divided into two groups for the experiment:

[0116] The first group of mice: The mice were randomly divided into three groups (n=4): (1) control group, (2) AIH group (1.5 mg / kg), and (3) FX (20 mg / kg) + AIH (1.5 mg / kg) group.

[0117] The second batch of mice: The mice were randomly divided into four groups (n=4): (1) AIH group (1.5 mg / kg), (2) 3-MA (30 mg / Kg) + AIH (1.5 mg / kg) group, (3) FX (20 mg / kg) + AIH (1.5 mg / kg) group, (4) 3-MA (30 mg / Kg) + FX (20 mg / kg) + AIH (1.5 mg / kg) group.

[0118] 2.2 Establishment of experimental autoimmune hepatitis mouse model and administration method

[0119] To construct an S100-induced autoimmune hepatitis (AIH) mouse model, 0.5 mL of liver S100 antigen emulsion (emulsified with 0.5 mL of complete Freund's adjuvant) was intraperitoneally injected on days 0 and 7. The control group was injected with 1 mL of sterile normal saline (NS). To evaluate the protective effect of fucoxanthin (FX), mice in the AIH+FX group received the same dose of S100 antigen injection on days 0 and 7, and 20 mg / kg of fucoxanthin was gavaged every other day starting from day 7.

[0120] To explore whether FX exerts its protective effect by regulating ferroptosis through mitochondrial autophagy, another group of experiments adopted the same AIH and FX treatment regimen, and the AIH+3-MA group and the AIH+3-MA+FX group were intraperitoneally injected with 3-MA every day starting from the 21st day.

[0121] 2.3 Sample collection

[0122] On the 28th day, the mice were anesthetized with sodium pentobarbital solution. After anesthesia, blood was collected from the heart, with a blood volume of about 500 to 1000 Ul. The mice were then fixed on their backs, the hair was shaved, and the abdomen and chest area were disinfected with 75% alcohol. After opening the abdomen, a 20 ml syringe (25 needle) was inserted into the left ventricle of the mouse, and pre-cooled PBS was injected for perfusion. At the same time, the right atrial appendage was cut off. After the liver was observed to have completely turned white, the liver tissue was separated and washed with 4°C PBS buffer. 1cm×1cm right lobe liver tissue was selected and fixed in 4% paraformaldehyde for later use; the remaining tissues were quickly frozen in liquid nitrogen and stored at -80°C.

[0123] 3. Liver Pathology Testing

[0124] 3.1 Hematoxylin & Eosin (HE) staining

[0125] 1) Fresh tissues were fixed in 4% paraformaldehyde for 48 h, dehydrated in 75%, 85%, 95%, and 100% ethanol solutions, and then embedded in paraffin. 4 μm paraffin sections were prepared using a paraffin slicer.

[0126] 2) Baking: Place the paraffin sections in a 60°C oven and bake for 1.5 hours.

[0127] 3) Dewaxing and hydration: Place the sections in xylene for 110 min - xylene II for 10 min - anhydrous ethanol for 5 min - 95% ethanol for 5 min - 85% ethanol for 5 min - 75% ethanol for 5 min - distilled water for 5 min.

[0128] 4) Hematoxylin staining of cell nuclei: Immerse the sections in hematoxylin staining solution for 5 minutes, rinse with tap water, immerse in 1% hydrochloric acid ethanol for 5 seconds, rinse with tap water for 2 minutes, put in 1% ammonia water to turn blue, and rinse with running water for 5 minutes.

[0129] 5) Eosin staining of cytoplasm: Immerse the sections in eosin staining solution for 3 minutes and rinse with tap water for 5 minutes.

[0130] 6) Dehydration and sealing: Place the stained sections in 75%, 85%, 95%, and 100% ethanol solutions in turn for 5 minutes each time; then place the sections in xylene I and xylene I in turn for 5 minutes each time. After the sections are slightly dried, seal them with neutral resin and place them under a microscope for observation and photography after they are naturally dried.

[0131] 4. Experimental testing

[0132] 4.1 Peripheral blood transaminase detection

[0133] After centrifugation at 3000 rpm for 10 min at 4°C, serum was separated for subsequent analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by an automatic biochemical analyzer (AU5800, Beckman Coulter, USA).

[0134] 4.2 IgG level detection

[0135] (1) Sample preparation

[0136] Bring mouse serum samples stored at -80°C to room temperature (25°C ± 3°C). 1× Wash Solution: Prepare with 20× Concentrated Wash Solution. 1× Detection Buffer: Prepare with 10× Concentrated Detection Buffer. Detection Antibody: Dilute 1:100 with 1× Detection Buffer. Serum samples need to be diluted 10,000 times.

[0137] (2) Draw a standard curve

[0138] Centrifuge the standard sample before opening the cover, re-dissolve the standard sample with distilled water, and vortex gently to mix it thoroughly. Take 230μL of the standard sample and add 230μL of 1× detection buffer, mix thoroughly to make the stock solution with a concentration of 400ng / mL. Based on the stock solution, dilute with 1× detection buffer to prepare solutions with concentration gradients of 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL, and 0ng / mL. Add 100μL of the standard sample to each standard well.

[0139] (3) Detection

[0140] ① Soak the microplate: Add 300 μL of 1× washing solution and soak for 30 seconds. Then discard the washing solution and pat the microplate dry on absorbent filter paper.

[0141] ② Sample addition: Add 90 μL 1× detection buffer and 10 μL pre-diluted sample to the sample well. Add 100 μL 1× detection buffer to the blank well.

[0142] ③ Incubation: Seal the plate with a sealing film. Shake at 100-300 rpm and incubate at room temperature (25℃±3℃) for 2 hours.

[0143] ④ Washing: Aspirate the liquid in the wells, add 300 μL of washing solution to each well, and wash 6 times.

[0144] ⑤ Add detection antibody: Add 100 μL of diluted antibody (1:100 dilution) to each well.

[0145] ⑥ Incubation: Seal the plate with a new sealing film. Oscillate at 100-300 rpm and incubate at room temperature (25℃±3℃) for 1 hour.

[0146] ⑦Washing: Repeat step ④.

[0147] ⑧ Add substrate for color development: Add 100 μL of color development substrate TMB to each well under light-proof conditions and incubate at room temperature (25°C ± 3°C) for 5-30 min.

[0148] ⑨Add stop solution: Add 100μL of stop solution to each well. The color will change from blue to yellow.

[0149] ⑩ Detection reading: Use a microplate reader to detect. Measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 570nm or 630nm. Then subtract the OD value of 570nm or 630nm from the OD value of 450nm for calibration.

[0150] 4.3 GSH detection

[0151] The micro-reduced glutathione (GSH) assay kit of Nanjing Jiancheng Bioengineering Institute was used to detect the tissue cells of each group of mice. The specific steps are as follows:

[0152] ① Sample preparation

[0153] Tissue sample processing: Take the homogenized tissue sample, mix it in an ice bath, take 0.1 ml of the supernatant, add 0.1 ml of reagent 1, mix it, centrifuge it at 3500 rpm and 4°C for 10 minutes, and take the supernatant for testing;

[0154] ②Measurement

[0155] The operation is as follows: mix well, let stand for 5 minutes, and measure the absorbance value of each well at 405 nm using an enzyme reader (measurement wavelength: 405 nm).

[0156]

[0157] ③GSH calculation formula

[0158]

[0159] 4.4 MDA detection

[0160] The lipid oxidation (MDA) detection kit of Nanjing Jiancheng was used to detect the tissue cells of each group of mice obtained in the above 2.3, and the MDA content (μmol / mg tissue) per unit weight of the cell tissue in each group of two batches of mice was measured, and the steps included:

[0161] ①Sample preparation

[0162] 1) Take 50 mg of tissue and add it to 450 uL of PBS, and grind the tissue with a tissue homogenizer;

[0163] 2) After homogenization, centrifuge at 2500 r / min for 10 min and take the supernatant for subsequent determination.

[0164] 3) For tissue samples, the protein concentration was determined by BCA after sample preparation, so as to facilitate the subsequent calculation of the MDA content per unit protein weight of tissue.

[0165] ② Preparation of the kit

[0166] 1) Preparation of Reagent 2: Take 2 mL of Reagent 2 in the kit and add it to 56.67 mL of distilled water and mix well. Preparation of Reagent 3: Add 60 mL of distilled water to Powder 3, heat to 90-100°C until fully dissolved, then add distilled water to 60 mL, and then add 60 mL of glacial acetic acid.

[0167] 2) MDA detection operation table:

[0168]

[0169] 3) Cover the centrifuge tube, poke a small hole in the cover with a needle, mix well with a vortex mixer, place in a 95°C water bath for 40 min, take out and cool with running water, then centrifuge at 4000 r / min for 10 min.

[0170] 4) Pipette 200uL of supernatant into a 96-well plate and read at 532nm.

[0171] 5) Calculation formula of MDA content in tissue:

[0172]

[0173] 4.5 Transmission Electron Microscopy (TEM)

[0174] Liver tissue and cells were fixed in 2.5% glutaraldehyde solution, and then fixed with 1% osmium tetroxide solution. After fixation, the samples were dehydrated by gradient concentrations of ethanol and acetone, and then the dehydrated tissue was immersed in a mixture of epoxy resin and propylene oxide and embedded at room temperature for 24 hours. Then, ultrathin sections were prepared and stained with lead citrate and uranyl acetate. Finally, mitochondrial morphology was observed using a transmission electron microscope (HT7800, HITACHI).

[0175] 4.6 Quantitative real time polymerase chain reaction (qRT-PCR)

[0176] ① Primer design and synthesis

[0177]

[0178] ② RNA extraction

[0179] 1) Weigh 50 mg of tissue into a 1.5 ml centrifuge tube, add 1 ml RNAiso Plus, and homogenize thoroughly on ice.

[0180] 2) Add 200uL of chloroform and shake vigorously for 15 seconds until all components are completely mixed and no longer separated, then let stand at room temperature for 5 minutes. Then, centrifuge at 12,000r / min at 4°C for 15 minutes.

[0181] 3) Carefully aspirate the upper transparent liquid (try to avoid aspirating the white film in the middle), then add an equal volume of pre-cooled isopropanol, invert and mix 10 times, and let stand at room temperature for 10 minutes. Finally, centrifuge at 12,000r / min at 4°C for 10 minutes.

[0182] 4) Discard the supernatant, add 1 ml of pre-cooled 75% ethanol (prepared with DEPC water), mix well (to make the precipitate float), and then centrifuge at 7500g and 4°C for 5 minutes to wash the precipitate.

[0183] 5) Aspirate and discard the supernatant, open the tube cap, and dry for 3-5 minutes until no ethanol remains.

[0184] 6) Depending on the amount of RNA, add an appropriate amount of DEPC water (usually 20-40 μl) to dissolve it, and repeatedly pipette until it is fully dissolved.

[0185] 7) RNA concentration and purity determination: Take 1 μL RNA, determine the concentration using Nanodrop 2000, and dilute the RNA to 200-1000 ng / μL with DEPC water. If the A260 / A280 ratio is between 1.9-2.1, the purity is high and can be used for subsequent experiments.

[0186] ③ RNA reverse transcription into cDNA

[0187] 1) Genomic DNA removal:

[0188]

[0189]

[0190] Mix by gently pipetting. Incubate at 42℃ for 2min.

[0191] 2) Prepare the reverse transcription reaction system:

[0192]

[0193] 3) Perform reverse transcription reaction

[0194] Reaction conditions: 37°C, 15 min for reverse transcription; 85°C, 5 s to inactivate reverse transcriptase.

[0195] ④qRT-PCR

[0196] 1) Prepare the following reaction system on ice:

[0197]

[0198] 2) The reaction program is set as follows:

[0199]

[0200] 3) Relative quantitative analysis:

[0201] After the test is completed, the Ct value data of each sample is obtained. The expression level of GAPDH is used as the internal reference, and the PCR relative quantification method is adopted. According to the formula: ΔCt = Ct to be tested - Ct internal reference, ΔΔCt = ΔCt experimental group - ΔCt control group, mRNA relative expression = 2-ΔΔCt, the relative level of gene expression of mRNA in the experimental group compared with that in the control group is calculated.

[0202] 4.7 Western Blotting (WB)

[0203] The steps include the following:

[0204] ① Protein extraction

[0205] 1) Weigh 50 mg of tissue using an electronic balance and place it in a 1.5 mL centrifuge tube. Then add 1 mL of tissue protein extract containing protease inhibitors and / or phosphatase inhibitors and grind thoroughly on ice to homogenize.

[0206] 2) The samples were sonicated for 2 minutes and then placed on ice for 30 minutes.

[0207] 3) Finally, centrifuge at 12,000 rpm at 4°C for 20 minutes and collect the supernatant.

[0208] ②BCA method to determine protein concentration

[0209] The BCA protein concentration assay kit of Shanghai Bio-Tech Biotechnology Co., Ltd. was used to detect the protein concentration of each sample. The specific steps are as follows:

[0210] 1) According to the number of sample and standard wells, prepare BCA working solution with a ratio of 50:1 of reagent A to reagent B and mix thoroughly. At the same time, preheat the constant temperature incubator to 37°C in advance.

[0211] 2) Prepare protein standard using PBS according to the instructions, with a final concentration of 0.5 mg / ml.

[0212] 3) Prepare standard curve: Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of prepared standard to the standard wells of a 96-well plate, and then add PBS to make up to a total volume of 20 μL per well, so that the final concentrations are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively.

[0213] 4) Dilute the sample to be tested with PBS to 1 / 20 of the original concentration, take 20 μL of the diluted sample and add it to the sample wells of a 96-well plate, and set up 3 replicate wells for each sample.

[0214] 5) Add 200 μL of the pre-prepared BCA working solution to the standard wells and sample wells respectively, place the 96-well plate in a constant temperature incubator preheated to 37°C, and incubate for 30 minutes.

[0215] 6) Use an ELISA reader to measure the absorbance at a wavelength of 562 nm, calculate the protein concentration of the sample based on the standard curve, and finally convert the actual concentration based on the dilution multiple.

[0216] ③Preparation of sample protein

[0217] According to the protein concentration, add an appropriate amount of PBS, and add 1 / 5 of the total volume for 5×5x dual-color loading to make the sample protein concentration obtained in ② consistent. After mixing, transfer the mixture to an EP tube.

[0218] Subsequently, the mixture was heated to 100°C in a thermostatic metal bath and boiled for 10 minutes to denature the protein.

[0219] ④SDS-PAGE electrophoresis detection

[0220] 1) Fix the 10% concentration precast gel on the sample rack, put it into the electrophoresis tank, and pour in 1× electrophoresis solution so that the glass plate is completely immersed in the electrophoresis solution.

[0221] Electrophoresis fluid (1×) configuration:

[0222]

[0223] 2) Pull out the comb of the precast gel in running water. Next, use a vortexer to mix the protein sample and load it in sequence according to the standard of 10 μl / well; after loading, add 2 μl of protein marker on both sides.

[0224] 3) Cover the lid, making sure that the color of the electrode on the lid matches the color of the electrophoresis tank; then perform electrophoresis at 80V for 30 minutes until the protein markers show clear stratified bands, at which point the voltage can be increased to 120V.

[0225] 4) When the target protein region is clearly stratified, the electrophoresis can be terminated.

[0226] ⑤ Cutting and transferring to film

[0227] 1) Place the sponge pad and transfer filter paper in order from bottom to top, place them on the "sandwich" transfer clip, and soak them with 1× transfer solution.

[0228] Transfer buffer (1×) configuration:

[0229]

[0230]

[0231] 2) Prepare PVDF membrane and transfer buffer: Select a PVDF membrane of appropriate size according to the size and number of target bands. Activate it in methanol for 15 seconds and then transfer it to 1× transfer buffer.

[0232] 3) Transfer gel and cut membrane: Cut the separation gel of the target protein molecular weight range, and clamp the transfer membrane clamp in the following order from bottom to top: sponge pad, two layers of filter paper, separation gel where the target protein is located, PVDF membrane, two layers of filter paper, sponge pad.

[0233] 4) Install the transfer clip and ice bath: Clamp the transfer clip, fix it in the transfer tank, and place its edge in the pre-frozen ice brick. Then, pour in the pre-cooled 1× transfer solution.

[0234] 5) Transfer settings: Transfer at 300 mA and determine the transfer time based on the molecular weight of the target protein.

[0235] ⑥ Closed

[0236] After electroporation, the strips were rinsed once with TBST, and the PVDF membrane was transferred to a pre-prepared and thoroughly dissolved mixed protein blocking solution (using 5% skim milk), and blocked at room temperature for 1.5 to 2 hours on a shaker at 40 rpm.

[0237] 5% blocking solution preparation:

[0238]

[0239] ⑦Incubate primary and secondary antibodies

[0240] 1) Washing: After blocking, place the strips on a shaker (100 rpm) and wash with 1×TBST for 10 minutes. Repeat the operation 3 times.

[0241] 2) Primary antibody incubation: Transfer the PVDF membrane to an EP tube containing the primary antibody diluted in a certain ratio according to the instructions, place it on a shaker (30 rpm), and incubate it overnight at 4°C. The incubation time should be controlled within 12-16 hours.

[0242] 3) Wash again: After recovering the primary antibody, place the strips on a shaker (80 rpm) and wash with 1× TBST for 10 minutes. Repeat the operation 3 times.

[0243] 4) Secondary antibody incubation: Place the strips in the pre-prepared corresponding secondary antibody, place on a shaker (40 rpm) and shake slowly, and incubate at room temperature for 1 hour.

[0244] 5) Final washing: After recovering the secondary antibody, the strips were placed on a shaker (100 rpm) and washed with 1× TBST for 10 minutes. The operation was repeated 3 times.

[0245] ⑧On the machine (chemical development)

[0246] 1) Prepare ECL chemical developer (A solution: B solution = 1:1)

[0247] 2) The liquid on the PVDF membrane is dried, and a developer is added, and the membrane is developed on a gel imaging system analyzer, and an image is collected.

[0248] ⑨Band analysis

[0249] Image J was used to count the grayscale of the bands.

[0250] ⑩Statistical methods

[0251] Statistical analysis was performed using GraphPad Prism version 9.0 software. Mean ± standard error (SEM) was expressed. One-way analysis of variance (ANOVA) and Tukey post hoc test were performed. p < 0.05 (*) was considered statistically significant.

[0252] V. Experimental Results

[0253] 5.1. Fucoxanthin improves liver damage and ferroptosis in S100-induced autoimmune hepatitis.

[0254] To explore the potential role of C66 in autoimmune hepatitis, we used 6-8 week old C57BL / 6L mice to construct an experimental autoimmune hepatitis model.

[0255] like Figure 1As shown in the figure, where: (A) Schematic diagram of the experimental procedure; (B) Representative H&E staining of liver tissue in each group, scale bar = 100 μm; (CE) Serum ALT, AST and IgG levels were measured by biochemical analyzer; (FH) PCR was used to detect the mRNA levels of Il1β, Tnfα and Il6 in the liver tissue of each group; the results were normalized to the expression of GAPDH protein, (IJ) The levels of MDA and GSH in liver tissue were detected by commercial detection kits; (KL) PCR was used to detect the mRNA levels of COX-2 and GPX4 in the liver tissue of each group; the results were normalized to the expression of GAPDH; (MO) The protein levels of COX-2 and GPX4 in liver tissue were analyzed using western blot; (P) Representative transmission electron microscopy images of mitochondria in liver tissue; red arrows indicate mitochondria, scale bar = 100 μm.

[0256] like Figure 1 As shown in (B), H&E staining was used to evaluate liver damage in the first group of mice. The H&E pathological results showed that compared with the normal control group, the livers of the AIH group mice had obvious inflammatory cell infiltration and pathological damage. Compared with the AIH group, the livers of the AIH+FX group mice had less inflammatory cell infiltration and pathological damage.

[0257] Plasma ALT, AST, and IgG levels in mice, such as Figure 1 As shown in (CE), compared with the normal control group, the serum ALT, AST and IgG levels in the AIH group were significantly increased, while the plasma ALT, AST and IgG levels in the AIH+FX group were significantly decreased. Figure 1 As shown in (FH), compared with the normal control group, the mRNA levels of inflammatory factors Tnfα, Il6 and Il1β in the liver of the AIH group were significantly increased, while treatment with fucoxanthin could reduce the expression of Tnfα, Il6 and Il1β mRNA. These results indicate that fucoxanthin improves liver damage in S100-induced autoimmune hepatitis.

[0258] To investigate whether FX can improve ferroptosis in S100-induced AIH, the concentrations of MDA and GSH in the first group of mice were measured. Figure 1 As shown in (IJ), FX improved the phenomenon of decreased GSH level and increased MDA level in the liver of AIH mice. Figure 1 As shown in (KO), compared with the normal group, in the AIH model, the mRNA and protein expressions of COX-2 were significantly increased, while the mRNA and protein expressions of GPX4 were significantly decreased. After FX treatment, these changes were effectively reversed. Figure 1As shown in (P), TEM observation further confirmed that the morphology of liver mitochondria in AIH mice was significantly abnormal, including mitochondrial cristae fracture, matrix inhomogeneity, and endoplasmic reticulum swelling and fracture, and FX treatment could significantly improve these abnormalities, and at the same time, double membranes appeared around some damaged mitochondria. It can be seen that fucoxanthin can improve liver damage and ferroptosis in S100-induced autoimmune hepatitis.

[0259] FX promotes mitochondrial autophagy in S100-induced autoimmune hepatitis

[0260] To further verify the effect of FX on mitophagy in S100-induced autoimmune hepatitis, we used the above-mentioned WB experimental detection method to detect the expression levels of mitophagy-related indicators in S100-induced autoimmune hepatitis mice.

[0261] like Figure 2 As shown, wherein: (AE) protein levels of PARKIN, PINK1, P62 and LC3 in liver tissues were analyzed by western blot.

[0262] like Figure 2 As shown in (AE), compared with the normal group, the protein expressions of PINK1, PARKIN, LC3II / I and P62 were significantly upregulated in the AIH mouse model group. Compared with the AIH group, the expressions of PINK1, PARKIN and LC3II / I in the AIH+FX group were further increased, and the protein expression of P62 was decreased. The above results suggest that FX promotes mitochondrial autophagy in S100-induced autoimmune hepatitis, allowing damaged mitochondria to be cleared in a timely manner.

[0263] FX inhibits S100-induced ferroptosis in AIH by promoting mitochondrial autophagy

[0264] In order to further verify the above conclusion, mice from the second group were taken for in vivo recovery experiments.

[0265] like Figure 3As shown, where: (A) Representative H&E staining of liver tissues in each group, scale bar = 100 μm; (BD) Serum ALT, AST and IgG levels were determined by biochemical analyzer; (EG) PCR was used to detect the mRNA levels of Il1β, Tnfα and Il6 in the liver tissues of each group; the results were normalized to the expression of GAPDH protein; (HI) The levels of MDA and GSH in liver tissues were detected by commercial detection kits; (JK) PCR was used to detect the mRNA levels of COX-2 and GPX4 in the liver tissues of each group; the results were normalized to the expression of GAPDH; (LR) Western blot was used to analyze the protein levels of PARKIN, PINK1, P62, LC3, COX-2 and GPX4 in liver tissues; (S) Representative transmission electron microscopy images of mitochondria in liver tissues; red arrows indicate mitochondria, scale bar = 100 μm.

[0266] Histologically, such as Figure 3 As shown in (A), H&E results showed that compared with the AIH group, the infiltration of liver inflammatory cells in the AIH+3-MA group was more severe, and the pathological damage in the AIH+3-MA+FX group was significantly more severe than that in the AIH+FX group. Figure 3 As shown in (BD), the levels of ALT, AST, and IgG in the AIH+3-MA group were higher than those in the AIH group, and these indicators in the AIH+3-MA+FX group were higher than those in the AIH+FX group; Figure 3 As shown in (EG), the mRNA expression levels of inflammatory-related factors Il1β, Tnfα, and Il6 in the AIH+3-MA group were significantly higher than those in the AIH group, and these factors were also significantly increased in the AIH+3-MA+FX group compared with the AIH+FX group; these results confirmed that FX could alleviate S100-induced AIH liver injury through mitochondrial autophagy, but this protective effect was weakened when autophagy was inhibited.

[0267] To further confirm whether FX improves ferroptosis by regulating mitochondrial autophagy, we also continued to detect the levels of MDA and GSH in the second batch of mice. Figure 3 As shown in (HI), compared with the AIH group, the GSH level in the AIH+3-MA group decreased and the MDA level increased, and the administration of FX partially reversed this change. Figure 3 As shown in (JK), the qRT-PCR results showed that compared with the AIH group, the expression of COX-2 mRNA in the AIH+3-MA group was increased, and the expression level of Gpx4 mRNA was decreased. The expression of COX-2 and Gpx4 mRNA in the AIH+3-MA+FX group was improved to a certain extent compared with the AIH+3-MA group, and after FX treatment. Figure 3As shown in (LR), WB results showed that compared with the AIH group, the protein levels of PINK1, PARKIN, LC3II / I and GPX4 in the AIH+3-MA group were downregulated, while the protein levels of P62 and COX-2 were upregulated. On this basis, FX treatment could not completely reverse the changes in the above indicators. Figure 3 As shown in (S), the liver mitochondria of mice in the AIH+3-MA group were more severely damaged, manifested by the disappearance of mitochondrial cristae and swelling and rupture of the endoplasmic reticulum. The mitochondrial morphology was improved to a certain extent after FX treatment. The above results indicate that FX may improve S100-induced AIH-related ferroptosis in the AIH mouse model by regulating mitochondrial autophagy.

[0268] In summary, the studies have shown that FX can regulate and activate mitochondrial autophagy-mediated ferroptosis. Based on the above findings, FX is expected to become a potential candidate drug for the treatment and prevention of AIH.

Claims

1. Use of fucoxanthin in the preparation of drugs for treating and preventing autoimmune hepatitis.

2. The use according to claim 1, characterized in that: The fucoxanthin reduces liver damage and plays a liver-protective role by inhibiting ferroptosis and activating mitochondrial autophagy signaling pathways.

3. The use according to claim 2, characterized in that: The fucoxanthin can inhibit ferroptosis by reducing the generation of lipid peroxides, upregulating the expression of glutathione peroxidase 4 (GPX4), downregulating the expression of cyclooxygenase 2 (COX-2), and alleviating the mitochondrial morphological changes associated with ferroptosis.

4. The use according to claim 2, characterized in that: The fucoxanthin can maintain the integrity of mitochondria by enhancing mitophagy.

5. The use according to claim 2, characterized in that: The fucoxanthin inhibits ferroptosis by alleviating lipid peroxidation through regulating mitochondrial autophagy.

6. The use according to claim 1, characterized in that: The fucoxanthin is used as an active ingredient to prepare a pharmaceutical composition for activating mitochondrial autophagy and inhibiting ferroptosis.

7. The use according to any one of claims 1 to 6, characterized in that: The medicine is an oral preparation or an injection preparation.

8. The use according to claim 7, characterized in that: The oral preparation is any one of tablets, capsules or granules.

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