Application of natural carotenoid fucoxanthine in medicine for treating autoimmune hepatitis

Abstract

The invention relates to an application of natural carotenoid fucoxanthine in a medicine for treating autoimmune hepatitis, which is characterized in that in order to improve mitochondrial injury in tissue cells, mitochondrial injury of tissue cells is induced by S100, a plurality of groups of control groups are set, and detection such as ATP (adenosine triphosphate) content detection, transmission electron microscope detection, qRT-PCR (quantitative real-time polymerase chain reaction) detection, WB (western blot) detection and IF (interstitial factor) detection is carried out. According to data analysis, the fucoxanthine improves mitochondrial damage of S100-induced mouse autoimmune hepatitis by regulating endoplasmic reticulum-mitochondrial coupling. By adopting the technical scheme, the invention provides the application of the natural carotenoid fucoxanthine in the medicine for treating the autoimmune hepatitis.

Description

Technical Field

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

[0002] Autoimmune hepatitis (AIH) is a chronic progressive liver inflammatory disease mediated by T cells in genetically susceptible individuals induced by environmental factors. It is characterized by elevated serum transaminase levels, positive serum autoantibodies, hyperimmunoglobulinemia and interface hepatitis. If not promptly intervened, it will develop into cirrhosis and liver failure. The disease currently has an increasing incidence, the pathogenesis is still unclear, and there is no drug that can completely cure it clinically.

[0003] Fucoxanthin (FX) is a natural carotenoid found in seaweed. It has anti-inflammatory, antioxidant, anti-tumor, and anti-obesity biological activities, and can improve endoplasmic reticulum stress. It has been proven to have a therapeutic effect on fatty liver, liver fibrosis, liver cancer and other liver diseases through different mechanisms of action. However, its role in autoimmune hepatitis has not yet been studied.

[0004] Mitochondria in liver tissue not only provide energy for the metabolism of liver cells, but also widely participate in the process of liver cell damage and even apoptosis. Mitochondrial damage, namely mitochondrial dysfunction, is mainly manifested in changes in mitochondrial morphology and structure, reduced ATP synthesis, kinetic imbalance, excessive production of reactive oxygen species, and mtDNA damage. Among them, mitochondrial ATP synthesis disorder and consumption and abnormal mitochondrial Ca2+ homeostasis will accelerate liver cell damage, and mitochondrial fission is an early event of necrotic cell death. These mitochondrial dysfunctions are involved in the occurrence and development of various liver diseases including viral hepatitis, alcoholic fatty liver, non-alcoholic fatty liver and hepatocellular carcinoma. In recent years, studies have found that mitochondrial damage is also related to the pathogenesis of AIH. Eliminating mitochondrial damage or improving mitochondrial quality control mechanisms can restore mitochondrial homeostasis and benefit liver health.

[0005] Therefore, targeting mitochondrial damage is a promising new therapeutic strategy for patients with autoimmune hepatitis. Summary of the invention

[0006] Purpose of the present invention: In order to achieve the goal of improving mitochondrial damage to treat autoimmune hepatitis, the present invention provides a use of a natural carotenoid fucoxanthin in a drug for treating autoimmune hepatitis, which can reduce mitochondrial damage to improve autoimmune hepatitis.

[0007] On the one hand, the present invention provides a use of a natural carotenoid fucoxanthin in a drug for treating autoimmune hepatitis.

[0008] Furthermore, the treatment of autoimmune hepatitis is to improve mitochondrial damage in tissue cells.

[0009] Furthermore, the fucoxanthin improves mitochondrial damage by regulating endoplasmic reticulum-mitochondrial coupling.

[0010] Another aspect of the present invention provides a drug for treating autoimmune hepatitis, wherein the drug comprises fucoxanthin.

[0011] Furthermore, the drug is an oral preparation or an injectable preparation.

[0012] Furthermore, the oral preparation is any one of tablets, capsules or granules.

[0013] The present invention induces mitochondrial damage in tissue cells by S100, and uses ATP content detection, transmission electron microscopy, qRT-PCR, WB and IF detection. According to data analysis, the fucoxanthin improves the mitochondrial damage of S100-induced autoimmune hepatitis in mice by regulating endoplasmic reticulum-mitochondria coupling. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Fucoxanthin improves liver damage and mitochondrial damage in S100-induced autoimmune hepatitis, including: (A) Modeling diagram; (B) Liver H&E staining, scale bar is 100μm; (C) Serum IgG level diagram; (D) Serum ALT and AST level diagram. (E) Expression of liver pro-inflammatory factors TnfαmRNA and Il1βmRNA; (F) Protein expression of DRP1, MFN2, BAX and Bcl2 in mouse liver tissue; (G) WB analysis of DRP1, MFN2, BAX and Bcl2 protein quantitative analysis statistics in mouse liver tissue; (H) ATP content level in mouse liver tissue (I) Transmission electron microscopy observation of mouse liver tissue mitochondria;

[0015] Figure 2 and Figure 3 Fucoxanthin improves the endoplasmic reticulum stress of S100-induced autoimmune hepatitis, including: (A) protein expression of GRP78, CHOP, IP3R, GRP75 and VDAC1 in mouse liver tissue; (B) quantitative analysis of GRP78, CHOP, IP3R, GRP75 and VDAC1 proteins in mouse liver tissue by WB analysis; (C) IF analysis of the interaction between IP3R and GRP75 proteins in mouse liver tissue; (D) IF analysis of the interaction between GRP75 and VDAC1 proteins in mouse liver tissue; (E) transmission electron microscopy observation of endoplasmic reticulum in mouse liver tissue;

[0016] Figure 4 Fucoxanthin improves S100-induced liver injury by inhibiting endoplasmic reticulum stress, including: (A) H&E staining of liver, scale bar is 100μm; (B) Serum IgG level diagram. (C) Serum ALT and AST levels diagram; (D) Expression of liver pro-inflammatory factors Tnfα mRNA and Il1β mRNA;

[0017] Figure 5 and Figure 6 Fucoxanthin improves S100-induced mitochondrial damage by inhibiting endoplasmic reticulum stress, including: (A) Protein expression of GRP78, CHOP, IP3R, GRP75, VDAC1, DRP1, MFN2, BAX and Bcl2 in mouse liver tissue; (B) Quantitative analysis of GRP78, CHOP, IP3R, GRP75, VDAC1, DRP1, MFN2, BAX and Bcl2 in mouse liver tissue by WB analysis; (C) IF analysis of the interaction between IP3R and GRP75 proteins in mouse liver tissue; (D) IF analysis of the interaction between GRP75 and VDAC1 proteins in mouse liver tissue; (E) ATP content level in mouse liver tissue. (F) Transmission electron microscopy observation of endoplasmic reticulum and mitochondria in mouse liver tissue. DETAILED DESCRIPTION

[0018] The technical scheme in this embodiment will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] It should be noted that in the description of the present invention, all directional indications (such as up, down, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0020] In addition, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that they can be implemented by those skilled in the art. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0021] 1. Experimental Preparation

[0022] 1.1 Experimental animals

[0023] The animals used were 6-8-week-old C57BL / 6N male mice purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd. (Zhejiang, China), housed in a specific pathogen-free (SPF) grade environment with a constant temperature of 25±2°C, a relative humidity of 60±5%, and a 12-h light / dark cycle, fed with a standard rodent diet, and had free access to purified water.

[0024] The care and use of mice were strictly followed in accordance with the Guide for the Care and Use of Laboratory Animals established by the National Institutes of Health (NIH).

[0025] 1.2 Experimental instruments

[0026] Centrifuges of various speeds, Thermo Fisher, USA

[0027] Tissue dehydrator, Shanghai Leica Instruments Co., Ltd.

[0028] Biological tissue automatic embedding machine, SLEE, Germany

[0029] KD-P tissue slide machine, Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd.

[0030] Rotary microtome, Shanghai Leica Instruments Co., Ltd.

[0031] Oven, Shanghai Yuejin Medical Equipment Co., Ltd.

[0032] Slides and coverslips, Wuhan Saiweier Biotechnology Co., Ltd.

[0033] Microwave oven, China Midea Group

[0034] Vortex Instrument, Chilin Bell Laboratory Instruments

[0035] Constant temperature metal water bath, Shanghai Yuejin Instrument Co., Ltd.

[0036] Optical microscope, Olympus, Japan

[0037] Fluorescence microscope, KEYENCE (China) Co., Ltd.

[0038] Transmission electron microscope, Hitachi, Japan

[0039] Nanodrop 2000, Thermo Fisher Scientific, USA

[0040] High temperature and high pressure sterilizer, Systec, Germany

[0041] Tissue grinder, Wuhan Saiweier Biotechnology Co., Ltd.

[0042] Electronic balance, MTD, USA

[0043] Ice Maker, Scotsman, Italy

[0044] -80℃ ultra-low temperature refrigerator, Thermo Fisher, USA

[0045] Electrophoresis apparatus, membrane transfer apparatus, Bio-rad, USA

[0046] Shaker, Chilin Bell Laboratory Instruments

[0047] Gel imaging system, Bio-rad, USA

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

[0049] PCR amplifier, Bio-rad, USA

[0050] Real-time fluorescence quantitative PCR system, fully automatic biochemical analyzer from Thermo Fisher, USA, biological safety cabinet and clean bench from BeckmanCoulter, USA, constant temperature drying oven from Qingdao Haier Biomedical Co., Ltd., Shanghai Yuejin Instrument Co., Ltd.

[0051] Full wavelength microplate reader, various types of scissors and tweezers from Medix Laboratory Instruments Co., Ltd., various specifications of measuring cylinders and beakers from Shanghai Jinzhong Medical Instruments Co., Ltd., Beijing Bomei Technology Co., Ltd.

[0052] 1.3 Experimental reagents

[0053] Fucoxanthin, MCE, USA

[0054] Tunicamycin, Selleck Biotechnology Co., Ltd., USA Sulfonbutyl-β-cyclohexane, Shanghai MacLean Biochemical Technology Co., Ltd. PEG300, Selleck Biotechnology Co., Ltd., USA

[0055] Tween80, Selleck Biotechnology Co., Ltd., USA

[0056] DMEM / F12 cell culture medium, FBS fetal bovine serum from Wuhan Pronocell Life Science Co., Ltd., trypsin digestion solution from Wuhan Pronocell Life Science Co., Ltd., ITS liquid culture medium supplement from Suzhou Xinsaimei Biotechnology Co., Ltd., Sigma, USA

[0057] DMSO, Beijing Solarbio Technology Co., Ltd.

[0058] CCK8, MCE, USA

[0059] Cultured cell total protein extraction reagent, Wuhan Boster Biotechnology Co., Ltd. mammalian tissue protein extraction reagent, Wuhan Boster Biotechnology Co., Ltd. PMSF protease inhibitor, Shanghai Biyuntian Biotechnology Co., Ltd. Anti-GRP78 antibody, Wuhan Sanying Biotechnology Co., Ltd.

[0060] Anti-CHOP antibody, Hangzhou Huaan Biotechnology Co., Ltd. Anti-MFN2 antibody, Hangzhou Huaan Biotechnology Co., Ltd. Anti-DRP1 antibody, Hangzhou Huaan Biotechnology Co., Ltd. Anti-BAX antibody, CST, USA

[0061] Anti-BCL2 antibody, Hangzhou Huaan Biotechnology Co., Ltd. Anti-IP3R antibody, Hangzhou Huaan Biotechnology Co., Ltd. Anti-GRP75 antibody, Santa Clara, USA

[0062] Anti-VDAC1 antibody, Hangzhou Huaan Biotechnology Co., Ltd. Anti-GAPDH antibody, Wuhan Tri-Eagle Biotechnology Co., Ltd.

[0063] Skim milk powder, BD, USA

[0064] BSA, goat anti-rabbit secondary antibody from BioFroxx Biotechnology Co., Ltd., Germany, goat anti-mouse secondary antibody from Shanghai Yamei Biopharmaceutical Technology Co., Ltd., prestained protein marker from Shanghai Yamei Biopharmaceutical Technology Co., Ltd., Western primary antibody diluent from Thermo Fisher Scientific, USA, 5X Loding Buffer (DTT) from Shanghai Biyuntian Biotechnology Co., Ltd., methanol from Hangzhou Fude Biotechnology Co., Ltd., Wenzhou Xiang'an Chemical Co., Ltd., Zhejiang

[0065] Glycine, sodium dodecyl sulfate SDS from Shanghai Biotech Co., Ltd., tris(hydroxymethyl)aminomethane TRIS from Beijing Solarbio Technology Co., Ltd., Immobilon-P PVDF transfer membranes of various pore sizes from Beijing Solarbio Technology Co., Ltd., protein-free rapid blocking solution (1X) from Millipore Co., Ltd., USA, ultrasensitive chemiluminescent substrate (ECL) from Shanghai Biotech Co., Ltd., TBS buffer (20×) from advansta, USA, Western primary and secondary antibody removal solution from Beijing Solarbio Technology Co., Ltd., TRIzol from Shanghai Biotech Co., Ltd., Invitrogen, USA

[0066] 1-Bromo-3-chloropropane, Shanghai MacLean Biochemical Technology Co., Ltd. Isopropanol, Sinopharm Chemical Reagent Co., Ltd.

[0067] PCR reverse transcription kit, Takara, Japan

[0068] DEPC water, Shanghai Jierui Bioengineering Co., Ltd.

[0069] TB Premix Ex TaqTMII enzyme kit, Takara, Japan RNAiso Plus, Takara, Japan

[0070] Anhydrous ethanol, Zhejiang Wenzhou Xiang'an Chemical Co., Ltd. Various PCR primers, Shanghai Shenggong Bioengineering Co., Ltd.

[0071] Hematoxylin-eosin (H&E) permanent staining reagent, Beijing Solarbio Technology Co., Ltd. Xylene reagent, Sinopharm Chemical Reagent Co., Ltd.

[0072] Hydrochloric acid differentiation solution, American Saiye Biotechnology Group

[0073] Neutral gum sealing medium, Sinopharm Chemical Reagent Co., Ltd.

[0074] DAB colorimetric reagent, DAKO, Denmark

[0075] 4% paraformaldehyde, Beijing Solarbio Technology Co., Ltd.

[0076] Pentobarbital sodium, Shanghai Ciyu Chemical Technology Co., Ltd.

[0077] Permeabilization agent (Triton X-100), Beijing Solarbio Technology Co., Ltd.

[0078] Fluorescent secondary antibody, Wuhan Tri-Eagle Biotechnology Co., Ltd.

[0079] Electron microscopy fixative, Wuhan Saiweier Biotechnology Co., Ltd.

[0080] ATP content detection kit, Beijing Box Biotechnology Co., Ltd.

[0081] Cell culture plates of various specifications, Nest Company, USA

[0082] Syringes of various specifications, BD Company, USA

[0083] Centrifuge tubes of various types, Nest, USA

[0084] Reagent sealing film, Bemis Company, USA

[0085] 1.4 Preparation of FX suspension for intragastric administration

[0086] Prepare 20% SBE-β-CD solution in normal saline: add 2 g SBE-β-CD (sulfobutyl ether β-cyclodextrin) powder to normal saline and make up to 10 ml. Use an ultrasonic bath or ultrasonic oscillator to ensure full dissolution (4°C, store for 1 week).

[0087] Preparation of fucoxanthin suspension: Weigh the required amount of fucoxanthin powder sample, add appropriate amount of DMSO, and prepare a 12.5 mg / mL clear DMSO stock solution. Mix the stock solution with 20% SBE-β-CD saline solution at a ratio of 1:9, and use an ultrasonic bath or ultrasonic oscillator again to mix evenly to obtain a 1.25 mg / mL fucoxanthin suspension.

[0088] 1.5 Preparation of Tunicamycin(TM) Solution for Intraperitoneal Injection

[0089] 1) Preparation of stock solution: Weigh 2 mg of Tunicamycin using an electronic balance, add 1 ml of DMSO, and prepare a 2 mg / mL stock solution (-20°C, storage for 3 months).

[0090] 2) Preparation of working solution: Take 50 μl of DMSO stock solution prepared in 1), add 400 μl PEG300, mix and clarify, add 50 μl Tween 80, mix and clarify, add 500 μl sterile H2O, mix and clarify, and obtain 0.1 mg / mL Tunicamycin working solution.

[0091] 2. Construction of Mouse Experiment

[0092] 2.1 Experimental Grouping

[0093] The mice weighing 20-25 g prepared above were divided into two groups:

[0094] The first batch of mice were randomly divided into three experimental groups: (1) normal control group, (2) AIH group, and (3) AIH+FX group, with 5 mice in each group;

[0095] The second batch of mice were randomly divided into 4 experimental groups: (1) AIH group; (2) AIH+TM group; (3) AIH+FX group; (4) AIH+TM+FX group, with 5 mice in each group.

[0096] 2.2 Establishment of experimental autoimmune hepatitis mouse model and administration method

[0097] (1) The first batch of mice: Figure 1 As shown in A

[0098] 1) Extraction of allogeneic liver antigen S100: A healthy male C57BL / 6 mouse was randomly selected and fixed on a sterile operating platform after pentobarbital anesthesia. A cross incision was made on the mouse chest with sterilized ophthalmic scissors to fully expose the chest and abdominal cavities, and then the liver was perfused through the heart until the blood color of the liver disappeared. Then the liver was separated and the gallbladder was removed. The entire liver was rinsed in clean pre-cooled PBS, cut into small pieces, and ground into tissue homogenate with a tissue homogenizer and collected in a 50ml centrifuge tube. After centrifugation at 150g and 4℃ for 10 minutes to remove the cell nucleus, the supernatant was collected and placed in an ultracentrifuge at 100000g and 4℃ for 1h. The supernatant obtained was S100. After the S100 protein concentration was measured by the BCA method, sterile PBS was added to dilute it and the concentration required for injection was prepared. After the protein was filtered through a filter, an equal volume of Freund's adjuvant was added to fully grind and emulsify it for the construction of mouse autoimmune hepatitis.

[0099] 2) The mice in the AIH group and the AIH+FX group were immunized by intraperitoneal injection of the liver S100 antigen emulsified mixture described in (1) on day 0 (the first injection was counted as day 0) and day 7, respectively. The mice in the control group were injected with 1 mL of sterile saline (NS). From day 7 onwards, the mice in the AIH+FX group were gavaged with 20 mg / kg FX suspension every other day, and the AIH group and the control group were gavaged with an equal amount of 20% SBE-β-CD saline solution.

[0100] (2) The second batch of mice

[0101] Each group of mice was immunized by intraperitoneal injection of the liver S100 antigen emulsified mixture described in (1) on day 0 (the first injection was counted as day 0) and day 7. From day 7, mice in the AIH+FX group and the AIH+TM+FX group were gavaged with 20 mg / kg FX suspension every other day, and mice in the AIH group and the AIH+TM group were gavaged with an equal amount of 20% SBE-β-CD saline solution. On days 20 and 27, mice in the AIH+TM group and the AIH+TM+FX group were intraperitoneally injected with 0.5 mg / kg TM solution, and mice in the other groups were injected with an equal amount of saline.

[0102] 2.3 Sample collection

[0103] On the 28th day of mouse modeling, the mice were anesthetized with sodium pentobarbital solution. After anesthesia, the mice were fixed on their backs on the operating table, the chest and abdominal areas of the mice were cleaned with 75% alcohol, the laparotomy was performed to fully expose the chest and abdomen, and pre-cooled PBS was used for perfusion through the heart. After the liver was observed to be completely white, the gallbladder was removed, the liver tissue was separated, and washed with 4°C PBS buffer. The right lobe of the liver with a size of 1 cm×1 cm was fixed in 4% polyformaldehyde for later use; part of the liver tissue was taken with a puncture needle and placed in electron microscopy fixative for later use, and the remaining tissue was quickly frozen with liquid nitrogen and stored at -80°C for later use.

[0104] 3. Liver Pathology Testing

[0105] 3.1 Paraffin-embedded sections

[0106] The liver tissues of each group of mice obtained in 2.3 above were processed using environmentally friendly dewaxing solution, paraffin embedding machine and paraffin slicer produced by SLEE of Germany, and the steps included:

[0107] (1) Fixation: Place the tissue in 4% paraformaldehyde for more than 24 hours.

[0108] (2) Dehydration and transparency: Soak the tissue in 75% alcohol for 4 h → 85% alcohol for 2 h → 90% alcohol for 2 h → 95% alcohol for 1 h → anhydrous ethanol I for 30 min → anhydrous ethanol II for 30 min → xylene I for 5-10 min → xylene II for 5-10 min.

[0109] (3) Wax immersion: The transparentized tissue is gradually immersed in paraffin I for 1 hour → paraffin II for 1 hour → paraffin III for 1 hour.

[0110] (4) Embedding: The wax-soaked tissue block is placed in a mold filled with paraffin for embedding, and then placed on the cold table of the paraffin embedding machine to cool. After cooling, the wax block is trimmed.

[0111] (5) Sectioning: Use a paraffin slicer to slice the tissue with a thickness of 4 μL. Place the slices in a slide floater and pick them up with a slide. Then, place the slices in an oven preheated to 60 degrees Celsius for 1.5 hours to ensure that the slices are fully fixed.

[0112] 3.2 H&E staining

[0113] The hematoxylin-eosin (H&E) high-definition permanent staining kit of Beijing Solarbio Technology Co., Ltd. was used to stain the liver tissues of each group of mice obtained in the above 2.3, and electron microscopy was performed to collect images, and the steps included:

[0114] (1) Dewaxing: Soak the sections in environmentally friendly dewaxing solution I for 20 min → environmentally friendly dewaxing solution II for 20 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 75% alcohol for 5 min, and rinse with distilled water.

[0115] (2) Pretreatment: Treat the sections with high-definition constant stain pretreatment solution for 1 min.

[0116] (3) Hematoxylin staining: Place the sections in hematoxylin stain for 3-5 minutes, then rinse with tap water, differentiate with differentiation solution, rinse with distilled water, then bluing with bluing solution, and rinse with distilled water.

[0117] (4) Eosin staining: Dehydrate the sections in 95% alcohol for 1 min, then stain them in eosin solution for 15 s.

[0118] (5) Dehydration: The sections were placed in anhydrous ethanol I for 2 min → anhydrous ethanol II for 2 min → anhydrous ethanol III for 2 min → n-butanol I for 2 min → n-butanol II for 2 min → xylene I for 2 min → xylene II for 2 min until transparent.

[0119] (6) Sealing: Use neutral gum to seal the slides.

[0120] (7) Microscopic examination and photography: Use an electron microscope to perform microscopic examination and collect images.

[0121] The environmentally friendly dewaxing liquid I, environmentally friendly dewaxing liquid II, anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, n-butanol I, n-butanol II, xylene I, xylene II, etc. used in the above steps are all reagents in the hematoxylin-eosin (H&E) high-definition constant staining kit of Beijing Solarbio Technology Co., Ltd.

[0122] 4. Experimental testing

[0123] 4.1 Determination of peripheral blood transaminases

[0124] Blood was collected from the eyeballs of mice in each group, placed in a 1.5 ml centrifuge tube and allowed to stand for 1 hour, centrifuged at 3000 rpm for 15 minutes at room temperature, and the supernatant was collected and stored in a -80°C ultra-low temperature refrigerator. Subsequently, the levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) in the blood samples were detected using an automatic biochemical analyzer (AU5800).

[0125] 4.2 Determination of serum IgG

[0126] (1) Sample preparation: restore the required reagents and serum samples to room temperature. Prepare 1× wash solution with 20× concentrated wash solution, prepare 1× detection buffer with 10× concentrated detection buffer, and dilute the detection antibody with 1× detection buffer at 1:100. Dilute the serum sample 10,000 times.

[0127] (2) Draw a standard curve: Centrifuge the standard instantaneously, re-dissolve it with distilled water, and oscillate to mix it thoroughly. Take 230μl of the standard and add 230μl of 1× detection buffer to mix it thoroughly to prepare a stock solution with a concentration of 400ng / mL. Dilute the stock solution with 1× detection buffer to prepare solutions with a concentration gradient of 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL, and 0ng / mL. Add 100μl of standard to each standard well.

[0128] (3) Sample testing:

[0129] 1) Soaking the ELISA plate: Add 300 μl of 1× washing solution to each well for soaking. After 30 seconds, discard the washing solution and pat the microplate dry with absorbent filter paper.

[0130] 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.

[0131] 3) Incubation and washing: Seal the plate with a sealing film. Shake at 100-300 rpm and incubate at room temperature for 2 hours. Then discard the liquid in each well and add 300 μl of washing solution for washing. Repeat 6 times.

[0132] 4) Add detection antibody: Add 100 μl of diluted detection antibody to each well.

[0133] 5) Incubation and washing: Seal the plate with a new sealing film. Shake at 100-300 rpm and incubate at room temperature for 1 hour. Then discard the liquid in each well and add 300 μl of washing solution for washing. Repeat 6 times.

[0134] 6) Add substrate for color development: Add 100 μl of color development substrate TMB to each well and incubate at room temperature in the dark for 5-30 minutes.

[0135] 7) Add stop solution: Add 100 μl of stop solution to each well. Observe that the color changes from blue to yellow.

[0136] 8) Detection reading: Use a full-band microplate reader to detect. Determine the OD value of the maximum absorption wavelength of 450nm as A1, and the OD value of the reference wavelength of 570nm or 630nm as A2. ΔOD value = A1-A2, and then use ΔOD value for calibration.

[0137] 4.3 ATP detection

[0138] The ATP content detection kit of Beijing Box Biotechnology Co., Ltd. was used to detect the tissue cells of each group of mice obtained in 2.3 above, and the steps include:

[0139] (1) Extraction of ATP from mouse liver tissue: 0.1 g of mouse liver tissue from each group was taken, 1 ml of extraction solution was added, and the mixture was homogenized in an ice bath. The mixture was then centrifuged at 10,000 g for 10 min at 4°C. After centrifugation, 500 μl of the supernatant was transferred to another centrifuge tube, 500 μL of chloroform was added, the mixture was thoroughly shaken and mixed, and the mixture was centrifuged at 10,000 g for 5 min at 4°C. The supernatant was placed on ice for testing.

[0140] (2) Measurement steps

[0141] 1) Preheat the microplate reader for more than 30 minutes, adjust the wavelength to 340nm, and zero it with distilled water.

[0142] 2) Prepare standard dilutions: dilute 20 μmol / mL ATP standard solution with distilled water to 2.5, 2.0, 1.0, 0.5, 0.25, and 0.125 μmol / mL, which are standard dilutions.

[0143] 3) Prepare the reagent 4 application solution (prepared immediately before use): prepare it according to the volume ratio of reagent 4: distilled water = 1:9 according to the usage amount.

[0144] 4) Prepare the reagent VI application solution (prepared immediately before use): prepare it according to the volume ratio of reagent VI: distilled water = 1:14 based on the usage amount.

[0145] 5) Prepare the detection working solution (prepared before use): Prepare the working solution according to the volume ratio of Reagent 2: Reagent 3: Reagent 4 application solution: Reagent 5: Reagent 6 application solution = 10:10:1:4:1 according to the usage amount, and preheat it in a 25°C water bath for more than 10 minutes before use.

[0146] 6) Add the following reagents in sequence to the 96-well UV plate:

[0147]

[0148] 7) Determine absorbance: Mix all reagents thoroughly and start timing immediately, measure the absorbance at 340 nm at the 10th second, record it as A1 determination and A1 standard; react accurately for 180 seconds, measure the absorbance at 340 nm at the 190th second, record it as A2 determination and A2 standard; calculate ΔA determination = A2 determination - A1 determination, ΔA standard = A2 standard - A1 standard.

[0149] 8) Establish a standard curve: Use 2.5, 2.0, 1.0, 0.5, 0.25, and 0.125 μmol / mL standard dilution concentrations as the abscissa (x) and the corresponding ΔA standard as the ordinate (y) to obtain the standard equation y=kx+b. Substitute the ΔA measurement into the formula to obtain x (μmol / mL).

[0150] (3) Calculation of ATP content

[0151]

[0152] (V extract: the volume of extract added during the extraction process, 1mL; W: the mass of the sample 0.1g)

[0153] The ATP content (μmol / g) in the tissue cells of each group of mice was calculated according to the formula, wherein the reagents used above were all reagents in the ATP content detection kit of Beijing Box Biotechnology Co., Ltd.

[0154] 4.4 TEM sample preparation and observation

[0155] (1) Fixation: Place fresh mouse liver tissue in electron microscopy fixative for more than 6 hours.

[0156] (2) Rinsing: Transfer the tissue to a new EP tube, add 1 ml of PBS for rinsing, centrifuge for 5 min, and let stand for 15 min (repeat 3 times).

[0157] (3) Post-fixation and staining: Discard the liquid, add 1-2 drops of 1% osmic acid, and incubate at room temperature in the dark for 1 hour. Add PBS, centrifuge for 5 minutes, and let stand for 15 minutes. Add double distilled water, centrifuge for 5 minutes, and let stand for 10 minutes. Discard the liquid, add 1-2 drops of uranyl acetate, and incubate at room temperature in the dark for 1-2 hours.

[0158] (4) Dehydration: Discard the uranyl acetate and perform gradient dehydration with acetone, 50%, 70%, 80%, 90% concentration once, 100% concentration twice, each time for 15-20 minutes. Centrifuge for 5 minutes and let stand for 15 minutes.

[0159] (5) Embedding and curing: Discard the liquid, add 1-2 drops of 1:1 embedding agent, place in a 37℃ oven, and avoid light for 1 hour. Discard the liquid, add 1-2 drops of 1:4 embedding agent, place in a 37℃ oven, and avoid light overnight. Transfer the sample to a new EP tube, add 1-2 drops of pure embedding agent, place in a 45℃ oven, and avoid light for 1 hour. Continue to embed and cure in a 45℃ oven, avoid light for 5 hours.

[0160] (6) Use an ultrathin microtome to slice the sample with a thickness of 70 nm.

[0161] (7) Transmission electron microscopy (Hitachi HT7800, Japan) was used to observe mitochondria and endoplasmic reticulum in liver tissue.

[0162] 4.5 Quantitative real time polymerase chain reaction (qRT-PCR)

[0163] (1) Design of synthetic primers

[0164]

[0165]

[0166] (2) Extraction of RNA

[0167] 1) Take the mouse tissues prepared in 2.3: weigh 30 mg of tissue using an electronic balance and put it into 1.5 ml of EP, add 1 ml of RNAiso Plus, homogenize thoroughly using a tissue homogenizer, and centrifuge at 12000g for 10 min at 4°C.

[0168] 2) Pipette the supernatant into a new EP tube and add 200 μl of chloroform. Shake vigorously for 15 seconds to fully mix the components. Then let it stand on ice for 5-10 minutes and centrifuge at 12,000 g for 10 minutes at 4°C.

[0169] 3) Carefully pipette 400 μl of the upper transparent aqueous layer into a new EP tube, and add an equal volume of pre-cooled isopropanol. Mix thoroughly and place on ice for 10 min. Finally, centrifuge at 12,000 g for 10 min at 4°C.

[0170] 4) Discard the supernatant and add 1 ml of pre-cooled 75% ethanol (prepared by diluting anhydrous ethanol with DEPC water). Mix thoroughly and centrifuge at 7500 g for 5 min at 4°C. Repeat 3 times to wash the precipitate.

[0171] 5) Aspirate and discard the supernatant, open the EP tube cap, and dry at room temperature for 3-5 minutes until no ethanol remains. The precipitate in the tube is RNA.

[0172] 6) Add 20-40 μl of DEPC water into the EP tube and blow repeatedly to fully dissolve it.

[0173] 7) Determine RNA concentration and purity (1.8-2.2): Pipette 1.5 μl RNA into Nanodrop2000 to test RNA concentration, then dilute RNA to an appropriate concentration (usually between 500-1000 ng / μl) with DEPC water.

[0174] (3) RNA reverse transcription into cDNA (Takara RR092A kit, 20 μl system)

[0175] 1) Removal of genomic DNA:

[0176]

[0177]

[0178] The mixture was gently pipetted to mix well and reverse transcription was performed at 42°C for 2 min.

[0179] 2) Prepare the reverse transcription reaction system:

[0180]

[0181] 3) Perform reverse transcription reaction

[0182] The reverse transcriptase was inactivated under the reaction conditions of 37°C, 10 min → 85°C, 5 s → 4°C, and end.

[0183] (4) qRT-PCR

[0184] 1) Prepare the reaction system on ice (20 μl):

[0185]

[0186] 2) Set up the reaction program:

[0187]

[0188]

[0189] 3) Relative quantitative analysis:

[0190] After the test is completed, the melting curve is observed to ensure that the primers are qualified. The Ct value of each sample is collected, and the expression level of Gapdh is used as the internal reference. 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 target gene expression is calculated.

[0191] 4.6 Western Blotting (WB Detection)

[0192] (1) Protein extraction: 50 mg of mouse liver tissue was weighed using an electronic balance and placed in 1.5 ml of EP, and 1 ml of tissue protein extract was added. The mixture was homogenized using a tissue homogenizer. The mixture was then centrifuged at 12,000 g for 15 min at 4°C and the supernatant was collected.

[0193] (2) Determination of protein concentration (BCA method)

[0194] 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:

[0195] 1) Prepare BCA working solution: Calculate the total amount of working solution required based on the number of standard and sample wells, prepare with a ratio of reagent A: reagent B = 50:1, and mix thoroughly.

[0196] 2) Prepare protein standard: add 1.2 ml of protein standard solution into a protein standard tube containing 30 mg BSA, fully dissolve to make 25 mg / mL protein standard solution, and then dilute it to 0.5 mg / mL with PBS.

[0197] 3) Prepare standard curve: add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the prepared standard to the standard wells in a 96-well plate, and add PBS to each well to make up to 20 μl volume, 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.

[0198] 4) Dilute the sample to be tested 50 times with PBS, and pipette 20 μl of the sample into the remaining wells of the 96-well plate, with 3 replicate wells set up for each sample.

[0199] 5) Add 200 μl of BCA working solution to each well and place the 96-well plate in a 37°C constant temperature incubator for 30 min.

[0200] 6) Use a full-band microplate reader to detect the absorbance value at a wavelength of A562nm, calculate the protein concentration of each sample based on the standard curve, and then convert it into the actual protein concentration according to the dilution ratio.

[0201] (3) Preparation of loading protein

[0202] According to the actual protein concentration measured in (2), add appropriate amount of PBS and 1 / 5 of the total volume of 5× dual-color loading to make the protein concentration of each sample consistent. After fully mixing, put it in a constant temperature metal bath and boil it at 100°C for 10 minutes to denature the protein.

[0203] (4) SDS-PAGE electrophoresis

[0204] 1) Prepare electrophoresis solution (1×):

[0205]

[0206] 2) Fix the 10% concentration of homemade gel in the electrophoresis tank, add 1× electrophoresis solution, immerse the glass plate completely in the electrophoresis solution, and carefully pull out the comb. Add 2μl of protein marker to the sample wells on both sides of the protein sample well for labeling, and fill it up to 10μl with 1× loading buffer. Then swirl and mix the sampled protein sample, and load 10μl into each well in turn.

[0207] 3) After loading, perform electrophoresis at a voltage of 10V for 10 minutes, then adjust the voltage to 80V and continue electrophoresis until the protein marker appears as clear stratified bands, at which point the voltage can be adjusted to 120V.

[0208] 4) Electrophoresis can be terminated when the protein band reaches the bottom of the glass plate.

[0209] (5) Transfer

[0210] 1) Prepare transfer solution (1×, pre-cooled):

[0211]

[0212] 2) Lay out the sponge pad and transfer filter paper in order, place them on the transfer clamp, and immerse them in 1× transfer solution.

[0213] 3) According to the size of the target band, cut the PVDF transfer membrane into appropriate size. After activation in methanol for 3 minutes, transfer it into 1× transfer buffer.

[0214] 4) Disassemble the glass plate, cut the gel in the corresponding area according to the molecular weight of the target protein, carefully transfer it to the transfer filter paper, and cover it with the activated labeled PVDF membrane.

[0215] 5) After clamping the transfer clip, fix it in the transfer tank and put frozen ice bricks around it. Then pour in the pre-cooled 1× transfer solution.

[0216] 6) Transfer the membrane at 250 mA constant current and ice bath. Set the transfer time according to the molecular weight of the target protein.

[0217] (6) Closed

[0218] 1) Prepare 5% blocking solution:

[0219]

[0220] After the transfer, the PVDF membrane was immersed in an incubation box containing 1×TBST, and the membrane was shaken rapidly at 110 rpm on a shaker for 10 min, after which the TBST was discarded and 5% skim milk was added for blocking. The membrane was then placed on a shaker and shaken slowly at 30 rpm for blocking at room temperature for 1.5 h.

[0221] (7) Incubation with primary and secondary antibodies

[0222] 1) Washing: Take out the blocked PVDF membrane and place it on a shaker. Wash it three times with 1×TBST, each time for 10 min, with a shaking speed of 110 rpm.

[0223] 2) Primary antibody incubation: Discard TBST and gently absorb excess TBST on the PVDF membrane with filter paper, place it in the pre-prepared primary antibody, place it on a shaker, and incubate overnight at 4°C and 30 rpm. The incubation time should be controlled within 12-16 hours.

[0224] 3) Wash again: After recovering the primary antibody, place the PVDF membrane on a shaker and wash it three times with 1×TBST, each time for 10 min, with a shaking speed of 110 rpm.

[0225] 4) Secondary antibody incubation: discard TBST, place the PVDF membrane in the pre-prepared secondary antibody, place it on a shaker and shake it slowly at 30 rpm, and incubate at room temperature for 1 hour.

[0226] 5) Final washing: After recovering the secondary antibody, the PVDF membrane was placed on a shaker and washed three times with 1× TBST, each time for 10 min, with a shaking speed of 110 rpm.

[0227] (8) Chemical development

[0228] ECL chemical developer (A solution: B solution = 1:1) was prepared according to the instructions. After the residual liquid was squeezed out of each strip membrane with filter paper, 200 μl of ECL chemical developer was evenly added to cover it, and the gel imaging system analyzer was used for development.

[0229] (9) Band analysis

[0230] Image J was used to count the grayscale of the bands.

[0231] 4.7 Immunofluorescence double staining

[0232] (1) Dewaxing and hydration: After the specimens were placed in a 60°C oven for 90 min, they were placed in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min, 75% ethanol for 5 min, double distilled water I for 5 min, and double distilled water II for 5 min.

[0233] (2) Antigen retrieval: Place the sections in a heat-resistant container containing sodium citrate solution (pH = 6.0), incubate at high temperature for 5 min, then at low temperature for 15 min. Remove the sections and cool them to room temperature. Wash them three times with PBS, each time for 5 min.

[0234] (3) Serum blocking: discard PBS, use a hydrophobic pen to limit the area, and block with 3% BSA at room temperature for 30 min.

[0235] (4) Primary antibody incubation: Discard the blocking solution, add two pre-configured primary antibodies from different species, shake and centrifuge, and incubate at 4°C overnight.

[0236] (5) Recover the primary antibody and wash three times with PBS, 5 min each time.

[0237] (6) Fluorescent secondary antibody incubation: discard PBS, add two pre-prepared fluorescent secondary antibodies from different species, incubate at room temperature for 1 hour, and then wash three times with PBS, each time for 5 minutes.

[0238] (7) Sealing: Discard PBS, seal the slides with DAPI, and cover with a coverslip.

[0239] (8) The samples were examined using a fluorescence microscope (Keyence) and images were collected.

[0240] 4.8 Statistical methods

[0241] GraphPad Prism 10.0 software was used for statistical analysis and drawing. For two groups of experimental data, two-tailed Student's t test was used for statistical comparison. For multiple groups of data, one-way analysis of variance (ANOVA) was used, and Tukey HSD test was used to further compare the groups. The experimental results are expressed as mean ± standard deviation, ns indicates no statistical significance, and p < 0.05 indicates statistically significant differences.

[0242] V. Experimental Results

[0243] 5.1. Fucoxanthin FX improves liver damage and mitochondrial damage in S100-induced autoimmune hepatitis

[0244] To explore the potential role of FX in autoimmune hepatitis, an experimental autoimmune hepatitis model was established using 6-8 week old C57BL / 6N mice.

[0245] like Figure 1 As shown in (B), H&E staining was used to evaluate liver injury 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 necrosis and inflammatory cell infiltration, while the liver necrosis and inflammatory cell infiltration of the mice after FX intervention were reduced.

[0246] In addition, the serum IgG levels of mice were examined, e.g. Figure 1 As shown in (C), compared with the normal control group, the serum IgG level of mice in the AIH group was increased, while the serum IgG level of mice in the AIH+FX group was decreased compared with the AIH group; the serum ALT and AST levels of mice were also detected. Figure 1 As shown in (D), compared with the normal control group, the serum ALT and AST levels of mice in the AIH group were significantly increased, while the increase in serum ALT and AST levels of mice after FX intervention was significantly alleviated; thus, it can be seen that fucoxanthin FX can improve S100-induced liver damage.

[0247] like Figure 1 As shown in (E), compared with the normal control group, the mRNA levels of pro-inflammatory factors Tnfα and Il1β in the liver of mice in the AIH group were significantly increased, while the addition of FX could reduce the expression of Tnfα and Il1β mRNA. These results further indicate that FX can improve the liver damage of S100-induced autoimmune hepatitis to a certain extent.

[0248] In order to explore whether FX can improve mitochondrial damage, the expression levels of DRP1, MFN2, BAX and Bcl2 proteins in the liver tissues of the first batch of mice were detected by WB. Figure 1 As shown in (FG), compared with the normal control group, the expression levels of DRP1 and BAX proteins in the AIH group were increased, and the expression levels of MFN2 and Bcl2 proteins were decreased. After FX intervention, the expression levels of DRP1 and BAX proteins decreased, and the expression levels of MFN2 and Bcl2 proteins increased. At the same time, the ATP content in the liver tissue of mice was measured, and the results were shown in Figure 1 As shown in (H), the ATP content in the liver tissue of mice in the AIH group was lower than that in the normal control group, while the ATP content in the liver tissue of mice in the AIH+FX group was higher than that in the AIH group. This shows that FX intervention can improve the damage of mitochondrial function.

[0249] In addition, the mitochondria of the first batch of mouse liver tissue were observed using transmission electron microscopy, and the results were as follows: Figure 1(I) shows that compared with the normal control group, the mitochondria in the liver tissue cells of AIH mice were swollen, the cristae were broken and disappeared, the matrix was uneven, and vacuolar electron-translucent areas appeared, while the above conditions were improved after FX intervention. This result shows that FX can improve the damage of mitochondrial morphology.

[0250] 5.2. FX improves endoplasmic reticulum stress in S100-induced autoimmune hepatitis

[0251] To further verify the effect of FX on ER stress in S100-induced autoimmune hepatitis, the expression levels of ER stress-related indicators in the first batch of mice were detected by WB.

[0252] like Figure 2 As shown in (AB), compared with the AIH group, the expression of endoplasmic reticulum stress proteins GRP78 and CHOP in the AIH+FX group mice decreased, the expression of endoplasmic reticulum-mitochondria coupling-related proteins IP3R and GRP75 decreased, and there was no significant change in VDAC1 protein. At the same time, the degree of interaction between endoplasmic reticulum-mitochondria coupling-related proteins IP3R-GRP75-VDAC1 was observed by immunofluorescence double staining. The results are shown in Figure 2 As shown in (CD), the interaction between IP3R-GRP75 and GRP75-VDAC1 was weakened in the AIH+FX group compared with the AIH group. In addition, the endoplasmic reticulum of the liver tissue of the first group of mice was observed using transmission electron microscopy. Figure 2 As shown in (E), the degree of ER swelling in liver tissue cells of mice in the AIH+FX group was significantly improved compared with that in the AIH group. The above results suggest that FX improves ER stress and ER-mitochondria coupling in the S100-induced autoimmune hepatitis mouse model.

[0253] 5.3. FX improves mitochondrial damage in S100-induced autoimmune hepatitis in mice by inhibiting ER stress and regulating ER-mitochondrial coupling.

[0254] In order to further verify the above conclusion, mice from the second group were taken for in vivo recovery experiments.

[0255] Histopathological aspects, such as Figure 3 As shown in (A), H&E results indicated that the extent of liver necrosis and inflammatory cell infiltration in mice in the AIH+TM group was higher than that in the AIH group, and the extent of liver necrosis and inflammatory cell infiltration in mice in the AIH+TM+FX group was higher than that in the AIH+FX group.

[0256] Serological aspects, such as Figure 3 As shown in (BC), the IgG, ALT and AST levels of mice in the AIH+TM group were higher than those in the AIH group, and the IgG, ALT and AST levels of mice in the AIH+TM+FX group were higher than those in the AIH+FX group. Figure 3 As shown in (D), the expression levels of Tnfα and Il1β mRNA in the AIH+TM group were higher than those in the AIH group, and the expression levels of Tnfα and Il1β mRNA in the AIH+TM+FX group were also higher than those in the AIH+FX group. Therefore, this part of the results confirmed that FX improved the liver damage of S100-induced autoimmune hepatitis at least in part by inhibiting endoplasmic reticulum stress.

[0257] To further confirm whether FX improves mitochondrial damage by inhibiting endoplasmic reticulum stress, the endoplasmic reticulum and mitochondria-related indicators of the second batch of mice were further tested:

[0258] like Figure 4 As shown in (AB), WB results showed that compared with the AIH group, the expression levels of GRP78, CHOP, IP3R, GRP75, DRP1, and BAX proteins in the AIH+TM group were increased, while the expression levels of MFN2 and Bcl2 proteins were decreased, and the expression level of VDAC1 protein did not change significantly. On this basis, the administration of FX could not completely reverse the changes in the expression levels of the above proteins caused by the endoplasmic reticulum stress agonist TM;

[0259] like Figure 4 As shown in (CD), the results of immunofluorescence double staining showed that the degree of interaction between IP3R-GRP75 and GRP75-VDAC1 increased in the AIH+TM group compared with the AIH group, and the degree of interaction between IP3R-GRP75 and GRP75-VDAC1 also increased in the AIH+TM+FX group compared with the AIH+FX group;

[0260] like Figure 4 As shown in (E), the ATP content in the liver tissue of mice in the AIH+TM group was lower than that in the AIH group, and the ATP content in the liver tissue of mice in the AIH+TM+FX group was also lower than that in the AIH+FX group.

[0261] like Figure 4 As shown in (F), transmission electron microscopy results indicated that compared with the AIH group, the endoplasmic reticulum of liver tissue cells in the AIH+TM group was swollen and the degree of mitochondrial damage was increased; while the degree of improvement of endoplasmic reticulum swelling and mitochondrial damage in the AIH+TM+FX group was lower than that in the AIH+FX group.

[0262] The above results indicate that FX may improve mitochondrial damage in S100-induced autoimmune hepatitis in mice by inhibiting ER stress and regulating ER-mitochondrial coupling.

[0263] In summary, our study found that fucoxanthin FX can improve mitochondrial damage in AIH by inhibiting ER stress and regulating ER-mitochondrial coupling, providing a new mechanism and possibility for the treatment of this disease.

Claims

1. Use of a natural carotenoid fucoxanthin in a drug for treating autoimmune hepatitis.

2. The use of a natural carotenoid fucoxanthin in a drug for treating autoimmune hepatitis according to claim 1, characterized in that: The treatment of autoimmune hepatitis is to improve mitochondrial damage in tissue cells.

3. The use of a natural carotenoid fucoxanthin in a drug for treating autoimmune hepatitis according to claim 2, characterized in that: The fucoxanthin improves mitochondrial damage by regulating endoplasmic reticulum-mitochondrial coupling mediated by endoplasmic reticulum stress.

4. A drug for treating autoimmune hepatitis, characterized in that: The drug includes fucoxanthin.

5. The drug for treating autoimmune hepatitis according to claim 4, characterized in that: The medicine is an oral preparation or an injection preparation.

6. The drug for treating autoimmune hepatitis according to claim 5, characterized in that: The oral preparation is any one of tablets, capsules or granules.

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