Application of sulforaphane in medicine for treating autoimmune hepatitis

Abstract

The invention relates to application of sulforaphane in research and development of a new medicine for treating autoimmune hepatitis, and provides application of SIRT1 / Akt serving as a target spot in inhibition of autoimmune hepatitis hepatocyte apoptosis. Through use of an SIRT1 inhibitor and detection and analysis of hepatic histological change, qRT-PCR, WB and the like, the invention confirms the application of SIRT1 / Akt as a target in treatment of the autoimmune hepatitis by sulforaphane, provides a new idea for treatment of the autoimmune hepatitis, and has important clinical application value.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of sulforaphane in medicines for treating autoimmune hepatitis. Background Art

[0002] Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease mediated by an abnormal autoimmune response, characterized by positive serum autoantibodies, high immunoglobulin G and / or γ-globulinemia, and interface hepatitis in liver histology. Individuals with a genetic susceptibility to AIH, under the promotion of endogenous and exogenous triggers, develop a self-persistent immune attack against hepatocytes, leading to the occurrence and development of the disease. AIH generally presents as a chronic, insidious onset, and current treatments mainly include glucocorticoids and immunosuppressants, and some patients are prone to drug resistance. Therefore, it is of great clinical significance to explore new treatment methods for AIH.

[0003] Sulforaphane (SFN) is an isothiocyanate phytochemical obtained by hydrolysis of glucosinolates by myrosinase in plants. It is abundant in cruciferous vegetables such as broccoli, cabbage and kale. SFN has multiple effects, such as antioxidant, anti-inflammatory, anti-diabetic, anti-cancer, neuroprotective and cardiovascular protective activities, and its drug toxicity to humans is low. Sulforaphane has an important influence on cell cycle, apoptosis, immune response and tumor development by regulating histone deacetylase gene expression. These effects make it a potential natural medicine with therapeutic effects on a variety of human diseases. Studies have found that sulforaphane can improve non-alcoholic fatty liver disease, inhibit liver fibrosis, improve liver insulin resistance, etc., and play a therapeutic role in liver diseases.

[0004] Apoptosis is a programmed cell death that depends on ATP and is characterized by cell shrinkage, chromatin condensation, DNA fragmentation into regular fragments, and cell membrane-wrapped apoptotic bodies, which are eventually engulfed by macrophages to reduce the leakage of cell contents and inflammatory response. Apoptosis is a complex biological process involving the regulation of multiple molecular mechanisms and signaling pathways. The normal progression of apoptosis is essential for maintaining tissue homeostasis and the balance of cell numbers during development. It has been reported that the occurrence and development of a variety of liver diseases are closely related to abnormal apoptosis of hepatocytes, including non-alcoholic fatty liver disease and autoimmune hepatitis.

[0005] SIRT1 (sirtuin-1) is an NAD+-dependent protein deacetylase that plays an important role in the regulation of apoptosis. More and more studies have found that SIRT1 can play a protective role in various diseases by inhibiting apoptosis. For example, in nucleus pulposus cells, SIRT1 can promote cell autophagy activity, inhibit apoptosis, and inhibit the degradation of the extracellular matrix caused by oxidative stress through the Akt pathway. SIRT1 regulates apoptosis through multiple mechanisms, including deacetylation of various gene proteins, thereby affecting the expression of apoptosis-related factors. Therefore, regulating hepatocyte apoptosis by SIRT1 / Akt is a new treatment strategy for autoimmune hepatitis. Summary of the invention

[0006] Purpose of the present invention: In order to explore a new method for treating autoimmune hepatitis, the present invention provides the role of sulforaphane in drugs for treating autoimmune hepatitis, which can alleviate autoimmune hepatitis by inhibiting cell apoptosis.

[0007] The invention illustrates that sulforaphane provides strong evidence for developing new drugs for treating autoimmune hepatitis.

[0008] Furthermore, the sulforaphane treats autoimmune hepatitis by inhibiting hepatocyte apoptosis.

[0009] Going deeper into the mechanism, sulforaphane inhibits apoptotic response by regulating the SIRT1 / Akt signaling pathway.

[0010] Beneficial effects:

[0011] The present invention uses S100 antigen protein to induce autoimmune hepatitis and apoptosis of liver tissue cells. Through the detection and analysis of liver histological changes, qRT-PCR and WB, it is found that sulforaphane can alleviate hepatocyte apoptosis in autoimmune hepatitis by regulating the SIRT1 / Akt signaling pathway, which provides a new idea for the treatment of autoimmune hepatitis and has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1Sulforaphane improves liver damage and hepatocyte apoptosis in S100-induced autoimmune hepatitis, including: (A) H&E staining of liver, scale bar is 100μm. (B) Serum ALT level. (C) Serum AST level. (D) ELISA method to measure the level of mouse serum IgG. (E) Expression of liver proinflammatory factor Il1β mRNA. (F) Expression of liver proinflammatory factor TNFα mRNA. (G) Western blot detection of the protein expression of Cleaved Caspase-3, Bcl-2, and Bax in mouse liver tissue. (H) Western blot quantitative statistical graph of Cleaved Caspase-3 protein in mouse liver tissue. (I) Western blot quantitative statistical graph of Bcl-2 protein in mouse liver tissue. (J) Western blot quantitative statistical graph of Bax protein in mouse liver tissue.

[0013] Figure 2 Sulforaphane regulates the SIRT1 / Akt pathway of S100-induced autoimmune hepatitis, including: (A) mRNA expression of liver SIRT1. (B) Western blot detection of SIRT1, Akt, and p-Akt protein expression in mouse liver tissue. (C) Western blot quantitative statistical graph of SIRT1 protein in mouse liver tissue. (D) Western blot quantitative statistical graph of p-Akt and Akt protein in mouse liver tissue.

[0014] Figure 3 Sulforaphane improves S100-induced liver injury by regulating SIRT1 / Akt pathway, including: (A) H&E staining of liver, scale bar is 100μm. (B) Serum ALT level. (C) Serum AST level. (D) ELISA determination of mouse serum IgG level. (E) Expression of liver proinflammatory factor Il1β mRNA. (F) Expression of liver proinflammatory factor TNFα mRNA.

[0015] Figure 4Sulforaphane improves S100-induced hepatocyte apoptosis by regulating the SIRT1 / Akt pathway, including: (A) Western blot detection of the protein expression of Cleaved Caspase-3, Bcl-2, and Bax in mouse liver tissue. (B) Western blot quantitative statistical graph of Cleaved Caspase-3 protein in mouse liver tissue. (C) Western blot quantitative statistical graph of Bcl-2 protein in mouse liver tissue. (D) Western blot quantitative statistical graph of Bax protein in mouse liver tissue. (E) mRNA expression of liver SIRT1. (F) Western blot detection of protein expression of SIRT1, Akt, and p-Akt in mouse liver tissue. (G) Western blot quantitative statistical graph of SIRT1 protein in mouse liver tissue. (H) Western blot quantitative statistical graph of p-Akt and Akt protein in mouse liver tissue.

[0016] Note: ns in the result graph means no statistical significance, *p<0.05 means the difference is statistically significant, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0017] 1. Experimental preparation and content

[0018] 1. Animal husbandry and model processing

[0019] 1.1 Preparation of experimental animals

[0020] All mice used were C57BL / 6J, male, 6-8 weeks old, weighing between 20±2g, and purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. The experimental mice were kept in an SPF-level barrier environment, which can maintain a sterile or near-sterile state to prevent the invasion of external microorganisms. The indoor temperature is usually maintained at 20-26°C, and the relative humidity is controlled at 40%-70% to ensure the comfort and health of the mice. A 12-hour day and night cycle is provided to simulate natural light conditions to ensure the normal biological rhythm of the mice. They are fed with autoclaved complete nutrient pellet feed and sterilized water, and the bedding is changed regularly and in time. The care and use of animals involved in the study are strictly carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals" approved by Wenzhou Medical University. This study was started 2 weeks after the mice adapted to the new SPF-level environment of the animal room.

[0021] 1.2 Preparation of S100 solution

[0022] (1) A male C57BL / 6J mouse was randomly selected for extraction of S100 liver antigen;

[0023] (2) giving the mice an intraperitoneal injection of sodium pentobarbital solution, and after anesthesia, fixing the limbs of the mice on the operating platform;

[0024] (3) Use ophthalmic scissors to make a cross-shaped laparotomy to clearly expose the mouse's abdominal and thoracic cavities, and perfuse the liver through the heart until the blood color of the liver disappears and the perfusion turns white;

[0025] (4) After removing the gallbladder, the liver was separated and taken out and placed in clean PBS pre-cooled on ice for subsequent experiments.

[0026] (5) Place the cleaned magnetic beads into EP tubes, add 700 μL of clean PBS to each EP tube, then cut the liver into pieces and place them evenly into each EP tube. Use a tissue grinder to fully grind the liver tissue into a tissue homogenate;

[0027] (6) Transfer the tissue homogenate in each EP tube to a 50 mL centrifuge tube and centrifuge at 150 g, 10 min, 4 °C to remove the cell nuclei;

[0028] (7) The supernatant obtained by centrifugation was divided equally into two medium-sized centrifuge tubes and centrifuged in an ultracentrifuge at 100,000 g (average speed), 1 h, and 4°C;

[0029] (8) The supernatant obtained by ultracentrifugation was aspirated into a 15 mL centrifuge tube, which is S100.

[0030] (9) The S100 protein concentration was determined using the BCA method. PBS was added as needed (required final concentration * required final volume / supernatant concentration = sample volume in the supernatant, PBS = final volume - sample volume in the supernatant) to make the S100 antigen protein concentration between 0.5 and 2 mg / mL. The protein was filtered and complete Freund's adjuvant was added at a volume ratio of 1:1. After sufficient grinding and emulsification, the mixture was loaded into a 2 mL syringe for later use.

[0031] 1.3 Preparation of sulforaphane working solution

[0032] Filter the sterile saline with a filter and slowly add 50 mg of sulforaphane solid powder until it is fully dissolved to obtain the corresponding concentration of SFN working solution. To prevent repeated freezing and thawing from causing the reagent to become ineffective, we dispensed the prepared SFN working solution into 1.5 mL sterile EP tubes and stored them in a -80°C refrigerator.

[0033] 1.4 Preparation of EX-527 working solution

[0034] 50 mg of EX-527 powdered drug was dissolved in 1 mL of DMSO as a stock solution (i.e., the stock solution concentration was 50 mg / mL) and stored in a -80°C refrigerator. In each experiment, EX-527 working solution (5% DMSO + 40% PEG300 + 5% Tween-80 + 50% sterile saline) was prepared in a certain proportion.

[0035] 1.5 Animal Experiment Grouping and Dosing Method

[0036] The first batch of mice were randomly divided into three groups, with n=4 mice in each group: (1) normal control group (Control); (2) AIH model group (S100); (3) sulforaphane treatment group (S100+SFN).

[0037] The second batch of mice was divided into 4 groups, with n = 4 mice in each group: (1) AIH model group (S100); (2) AIH model + SIRT1 inhibitor EX-527 group (S100 + EX-527); (3) sulforaphane treatment group (S100 + SFN); (4) sulforaphane treatment + SIRT1 inhibitor EX-527 group (S100 + EX-527 + SFN).

[0038] In the AIH group of the two batches of mice, each mouse was intraperitoneally injected with the prepared S100 liver-specific antigen mixture on day 0. Then, the S100 mixture was intraperitoneally injected again on day 7. After each injection, the mouse abdomen was gently rubbed to allow the S100 mixture to be fully mixed and absorbed in the abdominal cavity.

[0039] In the S100+SFN group of the two batches of mice, each mouse was intraperitoneally injected with the prepared S100 liver-specific antigen mixture on day 0 and day 7. And starting from day 7, sulforaphane working solution (7.5 mg / kg / d) was intraperitoneally injected every other day. After each injection, the abdomen of the mouse was gently rubbed to ensure that the drug was fully mixed and absorbed in the abdominal cavity.

[0040] In the S100+EX-527 group, each mouse was intraperitoneally injected with the prepared S100 liver-specific antigen mixture on days 0 and 7. In addition, EX-527 working solution (10 mg / kg / d) was intraperitoneally injected every other day starting from day 8. After each injection, the mouse abdomen was gently rubbed to ensure that the drug was fully mixed and absorbed in the abdominal cavity.

[0041] S100+EX-527+SFN, each mouse was intraperitoneally injected with the prepared S100 liver-specific antigen mixture on day 0 and day 7. And starting from the 8th day after the first administration of liver antigen S100, sulforaphane working solution (7.5mg / kg / d) was intraperitoneally injected every other day, and EX-527 working solution (10mg / kg / d) was intraperitoneally injected every other day starting from the 9th day. After each injection, the mouse abdomen should be gently rubbed to ensure that the drug is fully mixed and absorbed in the abdominal cavity.

[0042] 2. Experimental content

[0043] 2.1 Animal sample collection

[0044] On the 28th day after the first administration of liver antigen S100, the mice were anesthetized and killed to obtain relevant experimental samples. After the relevant experimental equipment was disinfected, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution, and blood was collected from the eye sockets. The mouse blood samples were collected in 1.5mL centrifuge tubes, placed in a 4℃ precooled centrifuge and centrifuged at 2000rpm for 15min, and the supernatant was retained; after centrifugation at 2000rpm for 10min, the supernatant was taken into a new centrifuge tube, which was the required serum and stored at -80℃ for subsequent determination of biochemical indicators. After completing the blood collection, the mouse limbs were fixed on a clean operating table, the mouse chest and abdomen were wiped with alcohol cotton balls, and a cross incision was made on the mouse chest with ophthalmic scissors to expose the contents of the chest and abdomen. Then, the pericardium was gently torn with tweezers to completely expose the heart, and the syringe filled with PBS buffer was carefully inserted into the left ventricle, and then a small incision was cut at the right atrial appendage, and the PBS buffer was pushed into the left ventricle for cardiac perfusion. After the liver turned completely white, the gallbladder was removed and the liver tissue was separated. After washing the liver tissue with pre-cooled PBS buffer, divide it into two parts: take a small piece of relatively intact mouse liver tissue and gently place it in a 5mL centrifuge tube containing 4% paraformaldehyde for fixation; the remaining liver tissue is evenly divided into cryopreservation tubes and stored in a -80℃ refrigerator for subsequent experiments.

[0045] 2.2 H&E staining of liver

[0046] The liver tissue fixed with 4% paraformaldehyde in 2.1 above was stained with hematoxylin-eosin (H&E), and images were collected by electron microscopy. The specific steps are as follows:

[0047] (1) Tissue embedding and sectioning

[0048] ① Sampling: After the mouse liver tissue is fixed in 4% paraformaldehyde for 24 hours, take out the tissue sample, rinse it with running water for 15 minutes, and put it into a dehydration box;

[0049] ② Dehydration: 75% ethanol (90 minutes) → 85% ethanol (120 minutes) → 90% ethanol (120 minutes) → 95% ethanol (120 minutes) → 100% ethanol I (90 minutes) → 100% ethanol II (60 minutes);

[0050] ③ Transparent: alcohol benzene (5-10 minutes) → xylene I (5-10 minutes) → xylene II (5-10 minutes);

[0051] ④ Wax immersion: immediately place the transparent tissue into the paraffin melted in the incubator: paraffin I (60 minutes) → paraffin II (60 minutes) → paraffin III (60 minutes);

[0052] ⑤Embedding: Place the wax-soaked tissue in the embedding machine for embedding: first put the melted wax into the embedding frame, take the tissue out of the dehydration box before the wax solidifies, put it into the embedding frame according to the requirements of the embedding surface and affix the corresponding label. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.

[0053] ⑥ Sectioning: Place the trimmed wax block on a paraffin slicer and slice it, with a thickness of about 4-6μm; note that the wax block needs to be pre-cooled before slicing to make the temperature of the wax and the tissue consistent and to make the wax block hard for easy slicing;

[0054] ⑦ Spreading: float the slices on 40℃ warm water on a spreader to flatten the tissue, pick up the tissue with a glass slide, and put it into a 60℃ oven to bake. After the water is dried and the wax is melted, take it out and store it at room temperature for later use.

[0055] (2) H&E staining

[0056] ① Dewaxing: xylene I (10 minutes) → xylene II (10 minutes) → 100% ethanol I (5 minutes) → 100% ethanol II (5 minutes) → 95% ethanol (5 minutes) → 90% ethanol (5 minutes) → 80% ethanol (5 minutes) → 70% ethanol (5 minutes) → wash with tap water for 2 minutes;

[0057] ②Hematoxylin staining: Hematoxylin staining (5-20 minutes) → Rinse with tap water (5 minutes) → Differentiation with differentiation solution (30 seconds) → Rinse with tap water (5 minutes) → Blueing with bluing solution (15-30 seconds) → Rinse with tap water (5 minutes);

[0058] ③ Eosin staining: 95% ethanol dehydration (1 minute) → place in eosin staining solution for 2 minutes;

[0059] ④ Dehydration and sealing: 95% ethanol I (1 minute) → 95% ethanol II (1 minute) → 100% ethanol I (1 minute) → 100% ethanol II (1 minute) → xylene I (1 minute) → xylene II (1 minute) → neutral resin sealing and observation under microscope.

[0060] 2.3 Determination of protein concentration (BCA method)

[0061] (1) Prepare BCA working solution

[0062] According to the required number of wells, prepare BCA working solution in a ratio of reagent A: reagent B = 50:1 (add 200 μL of reagent A × total number of wells, add 4 μL of reagent B × total number of wells), and mix the two.

[0063] (2) Preparation of protein standards

[0064] ① Add 1.2 mL of PBS buffer to the standard protein tube containing 30 mg BSA, mix thoroughly to dissolve, and obtain a 25 mg / mL protein standard solution. Aliquot and store in a -20°C refrigerator for later use;

[0065] ② Prepare a protein standard solution with a concentration of 0.5 mg / mL: Take 20 μL of the above-mentioned protein standard solution with a concentration of 25 mg / mL, add 980 μL PBS buffer, mix thoroughly, and dilute to obtain a protein standard solution with a final concentration of 0.5 mg / mL. Aliquot and store in a -20℃ refrigerator for later use.

[0066] (3) Determination of protein concentration

[0067] ① Select 8 wells in a 96-well plate for standard curve, add 20μL, 19μL, 18μL, 16μL, 12μL, 8μL, 4μL, 0μL of PBS buffer respectively, and then add 0μL, 1μL, 2μL, 4μL, 8μL, 12μL, 16μL, 20μL of 0.5mg / mL protein standard solution to make the final concentration of each well 0mg / mL, 0.025mg / mL, 0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL.

[0068] ② Dilute the sample to be tested 20 times with PBS buffer, set up 3 replicate wells for each sample, and add 20 μL of sample to each well.

[0069] ③ Add 200 μL of the BCA working solution prepared above to each well, and then incubate in a pre-heated 37°C constant temperature incubator for 30 minutes.

[0070] ④ Set the wavelength of the microplate reader to A562 nm, detect the absorbance, and then detect the sample protein concentration according to the standard curve.

[0071] 2.4 Western Blot Detection of Protein Expression

[0072] (1) Extraction of sample protein

[0073] Extraction of animal tissue protein

[0074] ① Take out the liver tissue previously stored in a -80℃ refrigerator and place it on ice. Cut 50mg of tissue on an electronic balance and rinse it 1-2 times with pre-cooled sterile PBS buffer, then place it in a labeled 1.5mL EP tube;

[0075] ② Add 1 mL of mammalian tissue protein extraction reagent and 10 μL of protease and phosphatase inhibitor mixture to the EP tube, add alcohol-disinfected magnetic beads, and then homogenize and grind in a certain mode in a tissue homogenizer;

[0076] ③Stand on ice for 30 minutes, centrifuge at 12000rpm, 15 minutes, 4℃. The liquid in the EP tube after centrifugation is divided into 3 layers. The clear liquid in the middle layer is the target protein;

[0077] ④After measuring the protein concentration by BCA, take the average value, use the Excel formula to input the concentration and calculate the amount of sample protein solution, 5× loading buffer and PBS to be added, so that the final protein concentration in each EP tube is consistent;

[0078] ⑤ Place the protein sample on ice, open the constant temperature metal bath and heat it to 100℃ in advance, centrifuge the protein stock solution at low speed, add PBS and 5× loading buffer to prepare the sample (add PBS→protein stock solution→5× loading buffer according to the measured concentration). After adding, boil at 100℃ for 15 minutes to denature, cool on ice, mix well with a vortex and centrifuge at low speed, and store the protein sample in a -80℃ refrigerator.

[0079] (2) SDS-PAGE electrophoresis

[0080] ① Preparation of 1× electrophoresis buffer: Tris-HCl 3.02g + glycine 18.89g + SDS 1.0g, dilute to 1L with ultrapure water;

[0081] ② Install the prefabricated glue, clamp the two plates and insert them to tighten them, align them to prevent glue leakage, and then install and fix them to the electrophoresis tank, pour in the electrophoresis liquid, fill the inner tank with electrophoresis liquid and remove the bubbles in the inner tank, and gently pull out the comb with both hands;

[0082] ③ Loading: Add 2 μL of protein marker to each well according to the pre-set order; mix with a vortex instrument and then centrifuge the protein sample, and load 20 μL or 30 μL of protein sample in order;

[0083] ④Electrophoresis: Put on the electrophoresis cover and connect the electrophoresis instrument (the red color of the electrode corresponds to the red color of the electrophoresis tank, and the black color of the electrode corresponds to the black color of the electrophoresis tank); first run the electrophoresis at 20V for 10 minutes, then adjust the voltage to 80V, and when the protein marker appears as clear stratified bands (about 30 minutes), increase the voltage to 120V to allow the bands to run to the bottom of the gel and then stop the electrophoresis.

[0084] (3) Transfer

[0085] ① Preparation of 1× transfer buffer: 200ml methanol + 3.02g Tris-HCl + 14.42g glycine, dilute to 1L with ultrapure water, and precool in a 4℃ refrigerator;

[0086] ② Preparation of PVDF membrane: Cut the PVDF membrane of corresponding size according to the length and width of the target band, activate it in methanol for 3 minutes, and then use tweezers to transfer it into the above-mentioned 1× transfer solution;

[0087] ③ Preparation of the "sandwich" transfer clip: Lay the transfer clip flat in a transfer container filled with 1× transfer solution, with the sponge pad and transfer filter paper from bottom to top;

[0088] ④ Installation of the "sandwich" transfer clip: Take out the precast gel after electrophoresis and put it into the transfer container to soak it. Use tools to pry open the glass plate and carefully transfer the gel to the filter paper. Then cut off the concentrated gel and the unwanted gel at the bottom according to the molecular weight of the target protein, and then cover the gel with the pre-methanol-activated PVDF membrane. Pay attention to exhaust the air between the membrane and the gel. Then, cover a layer of wet filter paper and sponge pad in turn, close the transfer clip to complete the preparation of the "sandwich" transfer clip. Carefully insert the clamped transfer clip into the red and black box transfer core (the black side of the transfer clip corresponds to the black side of the transfer core, and the white side of the transfer clip corresponds to the red side of the transfer core), then fix the red and black box transfer core in the transfer tank, put an ice box in the tank to lower the temperature, and pour in the pre-cooled 1× transfer solution, and finally cover the transfer tank (the red color of the electrode corresponds to the red color of the transfer tank, and the black color of the electrode corresponds to the black color of the transfer tank).

[0089] ⑤ Transfer settings: Place the transfer device in a basin filled with ice, and transfer the membrane at a constant current of 300mA in an ice bath. The transfer time is set according to the molecular weight of the target protein, as follows:

[0090]

[0091] (4) Closed

[0092] Pour the prepared blocking solution (5% skim milk: 1g milk + 20ml 1×TBST) into the incubation box, immerse the PVDF membrane in the blocking solution with the protein side facing up, and then incubate on a shaker at room temperature for 1.5 hours with the shaking speed set at 20-40rpm.

[0093] (5) Primary antibody incubation

[0094] Washing the membrane: After blocking, take out the strips and place them on a shaker at 100 rpm, wash with 1× TBST for 5 minutes, and repeat the operation 6 times;

[0095] Primary antibody incubation: Prepare the primary antibody at a certain dilution ratio in advance according to the instructions, absorb the excess 1×TBST on the strip with clean filter paper to prevent the primary antibody from being contaminated and diluted, place the strip in a 15ml centrifuge tube containing the primary antibody and incubate it at 4°C overnight (12-18 hours) to conduct antigen-antibody immune reaction between the specific antibody and the target protein.

[0096] (6) Secondary antibody incubation

[0097] Washing the membrane: After the primary antibody incubation is completed, the primary antibody is recovered and the strips are placed on a shaker at 100 rpm, and washed with 1× TBST for 5 minutes. Repeat the operation 6 times;

[0098] Primary antibody incubation: Prepare secondary antibody at a certain dilution ratio in advance according to the instructions. Use clean filter paper to absorb excess 1×TBST on the strips to prevent contamination and dilution of the secondary antibody. Place the strips in a 15 ml centrifuge tube containing the secondary antibody and incubate at room temperature for 1 hour.

[0099] Final membrane washing: After the secondary antibody incubation is completed, the secondary antibody is recovered and the strips are placed on a shaker at 100 rpm, washed with 1× TBST for 5 minutes, and repeated 6 times.

[0100] (7) On-machine development

[0101] Preparation of ECL exposure solution: The next step is strip exposure. Take 200 μl of each of ECL ultrasensitive exposure solution A and B (1:1) and mix thoroughly.

[0102] Take out the PVDF membrane, use clean filter paper to absorb the excess 1×TBST on the strip, carefully add ECL exposure solution to the protein surface of the PVDF membrane to avoid bubbles, and expose and develop on the machine according to the parameters set in advance.

[0103] (8) Band analysis

[0104] The grayscale values ​​of the bands were counted using Image J.

[0105] 2.5 Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR)

[0106] (1) RNA extraction

[0107] ① Sample preparation: First, label a 1.5ml EP tube, add 1ml of Trizol reagent to the EP tube, and place it on ice; take out the liver tissue from the -80℃ refrigerator, and weigh 50mg of the tissue with an electronic balance and put it into the corresponding EP tube just labeled, and then use a pre-cooled homogenizer to homogenize; transfer the homogenate to a centrifuge tube, let it stand at room temperature for 5 minutes, and then centrifuge it at 12000rpm for 15 minutes at 4℃, and carefully transfer the supernatant to a new enzyme-free EP tube;

[0108] ② Layering: Add 200μl of chloroform to the above EP tube (chloroform is easy to leak from the gun, pay attention to keep the EP tube as close to the chloroform bottle as possible and transfer the chloroform quickly), shake vigorously for 15 seconds to fully mix the chloroform and Trizol, let it stand for 5 minutes (chloroform on top, Trizol on the bottom), and then centrifuge at 12000rpm for 15 minutes at 4℃. At this time, the solution is divided into three layers: the lower organic layer, the middle layer is protein and DNA, and the top layer is the aqueous phase with dissolved RNA.

[0109] ③RNA precipitation: Carefully aspirate the supernatant (the tip of the gun should be close to the liquid surface and move down as the liquid surface moves down, "less is better than more"), then add an equal volume of isopropanol, invert up and down 20 times to mix, let stand on ice for 20 minutes, then centrifuge at 12000rpm for 10 minutes at 4℃, carefully discard the supernatant, and keep the white RNA precipitate at the bottom of the EP tube.

[0110] ④ RNA rinsing: prepare 75% ethanol (anhydrous ethanol: DEPC water = 3:1), add 1 mL of 75% ethanol, gently invert and mix to wash RNA, then centrifuge at 7500×g at 4°C for 10 minutes, pour out the upper layer of ethanol, repeat the operation 2-3 times, and centrifuge in the reverse direction each time; after the last empty tube centrifugation, carefully discard the supernatant, open the EP tube and turn it upside down, and dry it at room temperature for 10 minutes.

[0111] ⑤ Dissolve RNA: When the RNA precipitate is slightly moist and gel-like or translucent, add an appropriate amount of DEPC water (generally 20-40 μL), blow it repeatedly, and heat it at 55-60°C to promote the dissolution of RNA.

[0112] ⑥Measure the concentration: Clean the sample drop plate of the RNA concentration detection instrument with DEPC water and wipe it, then add 1.5μL of DEPC water to the sample well BLACK twice, so that the final result is close to 0; add 1.5μL of RNA solution to the sample well to detect its concentration, and dilute the RNA concentration to an appropriate level (between 200-1000ng / μL is more appropriate) with DEPC water, and the A260 / A280 ratio between 1.9-2.1 and the A260 / A230 ratio between 2.0-2.5 indicate that the RNA purity is good. If the RNA sample is not used immediately, it needs to be stored in a -80℃ refrigerator to avoid degradation.

[0113] (2) RNA reverse transcription

[0114] The samples must be kept on ice throughout the process to prevent degradation.

[0115] Takara RR092A kit to prepare 20μl system:

[0116] ① Reaction for removing genomic DNA: Prepare the reaction mixture on ice according to the following ingredients.

[0117]

[0118] Set reaction conditions: 42°C, 2 minutes

[0119] ② Reverse transcription reaction: Add 4 μl of 5X RT Premix to the reaction solution in ① on ice, mix gently and then immediately perform reverse transcription reaction:

[0120]

[0121] Set reaction conditions: 37℃, 10 minutes; 85℃, 5 seconds

[0122] If the synthesized cDNA needs to be stored for a long time, please store it in a -20℃ refrigerator.

[0123] (3) qRT-PCR

[0124] ① Primers are in dry powder state. Centrifuge at 4000 rpm for 1 minute, then add an appropriate amount of TE Buffer with low EDTA content to dissolve the primers to a concentration of 10 μM.

[0125] ② Prepare the following mixture in a qPCR tube:

[0126]

[0127] The amount of reverse transcription reaction solution (cDNA) added should not exceed 10% of the total volume of the PCR reaction solution.

[0128] ③Perform qPCR reaction according to the following conditions:

[0129]

[0130] ④Results calculation and analysis:

[0131] After the qRT-PCR test is completed, the CT value of the sample to be tested can be obtained, and GAPDH is used as the internal reference and calculated according to the formula of the relative quantitative method: ΔCT = CT 目的 -CT 内参 , ΔΔCT=ΔCT 实验组 -ΔCT 对照组 , the relative mRNA expression of the target gene in this sample = 2 -ΔΔCT 2.6 ELISA method for determination of serum IgG

[0132] (1) Sample preparation

[0133] Frozen serum samples were taken out of the refrigerator, thawed, and then diluted with 1× assay buffer.

[0134] (2) Preparation of standard dilution solution

[0135] Dilute the standard solution to 1× with double distilled water or deionized water.

[0136] (3) Preparation of washing liquid

[0137] Dilute the washing solution to 1× with double distilled water or deionized water.

[0138] (4) Detection

[0139] ① Soak the ELISA plate in 1× washing solution for 30 seconds, then take it out and pat it dry on thick absorbent paper;

[0140] ② Add 90 μL of 1× detection buffer and 10 μL of pre-diluted sample to the sample well. Add 100 μL of 1× detection buffer to the blank well;

[0141] ③ Seal the reaction wells with a sealing film and incubate at room temperature in the dark for 2 hours;

[0142] ④ Wash the plate 6 times, then pat dry on thick absorbent paper, then add 100 μl of diluted antibody, seal the reaction wells with sealing film, and incubate at room temperature in the dark for 1 hour;

[0143] ⑤ Wash the plate 6 times and pat dry on thick absorbent paper;

[0144] ⑥ Add 100 μl of color developer to each well and incubate at room temperature in the dark for 5-30 minutes;

[0145] ⑦ Add 100 μl of stop solution and wait until the color changes from blue to yellow.

[0146] ⑧ Use an enzyme-labeled instrument to measure the OD value of the maximum absorption wavelength of 450nm and the reference wavelength of 570nm or 630nm, and subtract the OD value of 450nm from the OD value of 570nm or 630nm for calibration.

[0147] 2. Experimental results

[0148] 2.1 Sulforaphane improves liver damage and hepatocyte apoptosis in S100-induced autoimmune hepatitis

[0149] In the first mouse model, we established an autoimmune hepatitis model by intraperitoneally injecting liver-specific antigen S100 into mice, and then intraperitoneally injected sulforaphane (SFN) to explore the effect of sulforaphane on autoimmune hepatitis. Figure 1 A shows that in H&E staining, compared with the normal control group, the liver tissue of the S100 model group mice showed obvious inflammatory cell infiltration. In the S100+SFN group, liver tissue necrosis and inflammatory cell infiltration were less than those in the S100 group.

[0150] At the same time, we tested the collected serum samples, such as Figure 1 As shown in (BC), compared with the normal control group, the serum ALT and AST levels of mice in the S100 group were significantly increased, while the serum ALT and AST levels of mice in the S100+SFN group were significantly decreased.

[0151] In addition, if Figure 1 As shown in (DF), ELISA and qRT-PCR were used to detect the serum IgG level and hepatocyte inflammatory factors (IL-1β, TNF-α) of mice. The results showed that these indicators were significantly increased in the S100 group, and sulforaphane could alleviate the increase of these indicators. It can be seen that sulforaphane improves liver damage and inflammatory response in S100-induced autoimmune hepatitis.

[0152] In order to explore whether sulforaphane can improve apoptosis in vivo, we used Western blot to detect the expression levels of apoptosis-related proteins in the liver. Figure 1 (GJ) The results showed that compared with the control group, the expression of pro-apoptotic proteins (CleavedCaspase-3, Bax) in the S100 group was increased, while the expression of anti-apoptotic protein (Bcl-2) was decreased.

[0153] In contrast, after the intervention of sulforaphane, the expression of pro-apoptotic proteins (Cleaved Caspase-3, Bax) decreased, while the expression of anti-apoptotic proteins (Bcl-2) increased. These results indicate that sulforaphane can improve hepatocyte apoptosis in S100-induced autoimmune hepatitis in mice to a certain extent.

[0154] 2.2 Sulforaphane regulates the SIRT1 / Akt pathway in S100-induced autoimmune hepatitis

[0155] In order to clarify whether SIRT1 / Akt plays a role in the treatment of autoimmune hepatitis with sulforaphane, we measured the expression of related indicators in the liver tissues of each group of mice by qRT-PCR and Western blot.

[0156] like Figure 2 As shown, qRT-PCR results showed that compared with the normal control group, the mRNA expression level of SIRT1 in the S100 group was decreased, and the mRNA expression level of SIRT1 in the S100+SFN group was increased.

[0157] The results of Western blot are similar to this, such as Figure 2 As shown in (BD), S100 modeling significantly decreased SIRT1 protein expression and p-Akt / Akt phosphorylation levels. Compared with the S100 group, the SIRT1 protein expression and p-Akt / Akt phosphorylation levels in the S100+SFN group increased. These results suggest that sulforaphane can activate the SIRT1 / Akt pathway in mice with autoimmune hepatitis.

[0158] 2.3 Sulforaphane improves S100-induced liver injury by regulating SIRT1 / Akt pathway

[0159] To further verify whether sulforaphane improves autoimmune hepatitis through the SIRT1 / Akt signaling pathway, we performed a recovery experiment. We used the SIRT1 inhibitor EX-527 to inhibit the SIRT1 / Akt pathway.

[0160] like Figure 3 As shown in A, the results of liver histology H&E staining experiments showed that the necrosis area and inflammatory cell infiltration of the liver tissue in the S100+EX-527 group were larger than those in the S100 group. At the same time, compared with the S100+SFN group, the necrosis area and inflammatory cell infiltration of the liver tissue in the S100+EX-527+SFN group were larger. This suggests that the addition of the SIRT1 inhibitor EX-527 will cause more severe damage to liver tissue.

[0161] like Figure 3 As shown in (BC), the ALT and AST levels in the S100+EX-527 group were higher than those in the S100 group. In addition, the serum ALT and AST levels in the S100+EX-527+SFN group were also higher than those in the S100+SFN group. This suggests that the SIRT1 inhibitor EX-527 can aggravate liver damage.

[0162] In addition, if Figure 3As shown in (DF), we used ELISA and RT-qPCR to detect the levels of serum IgG and hepatocyte inflammatory factors (IL-1β, TNFα) in mice. Compared with the S100 group, the levels of serum IgG and pro-inflammatory factors (IL-1β, TNF-α) in the S100+EX-527 group were significantly increased. The levels of serum IgG and pro-inflammatory factors (IL-1β, TNF-α) in the S100+EX-527+SFN group were significantly increased compared with the S100+SFN group. This suggests that the inhibition of SIRT1 increases the release of inflammatory mediators.

[0163] In summary, this part of the results verified that sulforaphane can improve liver damage in S100-induced autoimmune hepatitis in mice by activating the SIRT1 / Akt pathway.

[0164] 2.4 Sulforaphane improves S100-induced hepatocyte apoptosis by regulating SIRT1 / Akt pathway

[0165] In order to further explore whether sulforaphane can improve hepatocyte apoptosis in S100-induced autoimmune hepatitis mice by activating the SIRT1 / Akt pathway, we added the SIRT1 inhibitor EX-527 to the S100 model and tested related indicators. Figure 4 As shown in (AD), Western blot results confirmed that inhibition of SIRT1 increased hepatocyte apoptosis. The expression of pro-apoptotic proteins (Cleaved Caspase-3, Bax) in the liver tissue of mice in the S100+EX-527 group was increased compared with that in the S100 group, while the expression of anti-apoptotic protein (Bcl-2) was decreased compared with that in the S100 group. Compared with the S100+SFN group, the expression of pro-apoptotic proteins (Cleaved Caspase-3, Bax) in the S100+EX-527+SFN group was increased, while the expression of anti-apoptotic protein (Bcl-2) was decreased.

[0166] We further tested the related indicators of SIRT1 / Akt pathway. Figure 4 As shown in E, the addition of EX-527 inhibited the mRNA content of SIRT1, that is, the mRNA level of SIRT1 in the S100+EX-527 group was downregulated compared with the S100 group, and the mRNA level of SIRT1 in the S100+EX-527+SFN group was also lower than that in the S100+SFN group.

[0167] like Figure 4As shown in (FH), Western blot analysis of the results at the protein level also showed the same trend as above. The protein expression level of SIRT1 in the liver tissue of mice in the S100+EX-527 group was lower than that in the S100 group. Compared with the S100+SFN group, the protein expression level of SIRT1 in the liver tissue of the S100+EX-527+SFN group was reduced. In addition, the detection of Akt content and its phosphorylation level indicated that the p-Akt / Akt phosphorylation level in the S100+EX-527 group was lower than that in the S100 group. Compared with the S100+SFN group, the p-Akt / Akt phosphorylation level in the S100+EX-527+SFN group decreased.

[0168] The above results show that sulforaphane improves hepatocyte apoptosis in S100-induced autoimmune hepatitis in mice by activating the SIRT1 / Akt pathway.

Claims

1. Application of sulforaphane in drugs for the treatment of autoimmune hepatitis.

2. The use of sulforaphane in a drug for treating autoimmune hepatitis according to claim 1, characterized in that: The treatment of autoimmune hepatitis is to inhibit apoptosis of hepatocytes in tissue cells.

3. The use according to claim 2, characterized in that: The sulforaphane inhibits the apoptotic response in autoimmune hepatitis by regulating the SIRT1 / Akt signaling pathway.

Images