Curdlan fullerene derivative for treating autoimmune hepatitis and its preparation method and application

Abstract

The present invention discloses a curdlan fullerene derivative, its preparation method, and application. The curdlan fullerene derivative comprises curdlan and a fullerene derivative, wherein the curdlan and the fullerene are covalently synthesized by amidation. The curdlan is a water-soluble curdlan derivative, and the fullerene is a carboxylated fullerene derivative. The curdlan fullerene provided by the present invention can efficiently scavenge free radicals, protect cells, and reduce oxidative damage. The curdlan fullerene material of the present invention can be used to treat liver-related diseases such as autoimmune hepatitis, viral hepatitis, liver damage, cirrhosis, hepatic steatosis, and other liver diseases through intravenous administration, intraperitoneal administration, and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a curdlan fullerene derivative for treating autoimmune hepatitis, and a preparation method and application thereof. Background Art

[0002] Autoimmune hepatitis (AIH) is a progressive, immune-mediated inflammatory disease of the liver. Its typical pathological features are abnormal serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the presence of circulating autoantibodies, significantly elevated immunoglobulin G (IgG), and moderate to severe interstitial inflammation. In recent years, the incidence of AIH has been steadily increasing. Following onset, AIH is highly susceptible to progression to liver fibrosis, cirrhosis, and liver failure. 30%-50% of patients will still develop cirrhosis despite treatment, posing a serious threat to human health and safety.

[0003] Currently, there is no clinical treatment targeting the cause of AIH. Nonspecific immunosuppressive drugs are primarily used to induce disease remission and improve patients' clinical symptoms. First-line treatment is primarily with glucocorticoids (such as prednisone) alone or in combination with azathioprine. Although these immunosuppressants can significantly improve symptoms in the short term, 70% of patients will relapse after discontinuation, requiring lifelong immunosuppressive therapy for most patients. However, long-term use of immunosuppressants also presents a range of side effects, such as bone marrow suppression, increased risk of fractures, cataracts, blood system damage, and increased risk of tumors. Furthermore, some patients are tolerant to these immunosuppressants. Purine antagonists (mycophenolate mofetil) can be used as second-line drugs. However, controlled clinical trials are lacking, and no drug has been designated as a standard second-line treatment. Therefore, new methods to effectively control or even cure autoimmune hepatitis are still needed.

[0004] Fullerenes are the third allotrope of carbon, after diamond and graphite. They are the only cage-shaped pure carbon structures with a fixed molecular weight, and their properties can be modified both inside and outside the cage. Fullerenes' high stability and pure carbon structure give them excellent biocompatibility. Their high symmetry and unique conjugated electron cloud on their molecular surface can be used to efficiently quench excess free radicals, protect and repair cell damage, resist inflammatory responses, and regulate immunity. However, fullerenes themselves are insoluble in water, making them difficult to use directly in biological systems. Therefore, functionalizing fullerenes to improve their water solubility is a key issue that needs to be addressed. Common fullerene functionalization modifications include physical modification (coating with cyclodextrins, polymers, etc.) and chemical modification (chemically bonding amino, hydroxyl, carboxyl, and other active groups) to enhance the water solubility of fullerene materials. Natural polymers such as polysaccharides are widely used in nanomaterial modification due to their excellent biodegradability and bioactivity. Curdlan is a biodegradable 1,3-β-D glucan approved by the FDA as a food additive in 1996. It has good biocompatibility. Studies have found that natural curdlan also has immunomodulatory and anti-tumor activities. The hydroxyl group at position 6 of curdlan is more reactive than other positions, and other active groups can be introduced through a series of chemical reactions, making it easy to modify. Therefore, this project intends to use curdlan for the functional modification of fullerene materials, which can not only enhance the biocompatibility of fullerene materials, but also synergistically improve the effectiveness of fullerene materials in regulating immunity and inflammation.

[0005] Autoimmune hepatitis is both an autoimmune disease and a liver inflammatory disease, so its treatment can be approached from two aspects. In terms of immune diseases, fullerenes can regulate macrophage differentiation, regulate the Th1 / Th2 cell balance, and regulate the immune system response. Curdlan can bind to macrophages and stimulate immune cells to produce cytokines through the Dectin-1 signaling pathway. In terms of liver inflammation, fullerenes can efficiently remove excess ROS from liver cells, relieve liver cell oxidative stress, and resist inflammation. Therefore, the use of curdlan for the functional modification of fullerenes is expected to play an excellent role in autoimmune hepatitis. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a curdlan fullerene derivative with good biocompatibility and biosafety and a preparation method thereof.

[0007] In order to achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] A curdlan fullerene derivative is prepared from curdlan and a fullerene derivative, wherein the curdlan and the fullerene derivative are covalently linked through amidation.

[0009] The curdlan described in the present invention is a water-soluble curdlan derivative, including water-soluble curdlan derivatives modified by hydroxylation, amination, carboxylation or other methods, specifically a curdlan derivative modified by a water-soluble small molecule with terminal amino and alkyne groups through a click reaction.

[0010] According to a specific embodiment of the present invention, the curdlan is an amino-modified curdlan, and its preparation method comprises the following steps:

[0011] 1) Azide-modifying the C6 position of curdlan to synthesize azidated curdlan;

[0012] 2) According to the Click reaction mechanism, amino gellan was synthesized from azidated gellan and propargylated diglycolamine.

[0013] The fullerene derivatives used in the present invention are carboxylated fullerene derivatives, including fullerene derivatives linked to fullerenes via methods such as the Bingel reaction, the Prato reaction, and an amination reaction, specifically lysine fullerene derivatives, glycine-modified fullerene derivatives, and alanine-modified fullerene derivatives. They also include fullerene derivatives linked via non-covalent interactions, such as carboxylated polyethylene glycol-modified fullerene derivatives and carboxylated polyvinylpyrrolidone-modified fullerene derivatives.

[0014] The fullerenes used in the present invention include hollow fullerenes and endohedral metallofullerenes. The hollow fullerenes can be represented by C 2n , 30≤n≤60, specifically C 60 or C 70 The embedded metallofullerene can be represented as M@C 2n , 30≤n≤60, wherein M is selected from any one of Sc, Y and lanthanide metal elements (La-Lu), and specifically Gd@C 82 .

[0015] Furthermore, the molar ratio of the curdlan to the fullerene derivative of the present invention can be 1:(0.5-4). The degree of fullerene substitution in the curdlan fullerene synthesized at different ratios varies, ranging from 0.2-0.9, specifically 0.67. The hydrated particle size of the curdlan fullerene derivative provided by the present invention is 50-200 nm, and the potential is -10 mV to -40 mV.

[0016] The curdlan fullerene proposed by the present invention can efficiently scavenge free radicals, protect cells, and reduce oxidative damage. The curdlan fullerene proposed by the present invention is more enriched in macrophages.

[0017] The present invention also provides a method for preparing the curdlan fullerene derivative.

[0018] The preparation method of the curdlan fullerene derivative provided by the present invention comprises the following steps: under the activation of EDC and NHS, the amino curdlan and the carboxylated fullerene derivative are subjected to an amide reaction to obtain the curdlan fullerene derivative.

[0019] First, the carboxylated fullerene derivative is dissolved in a sodium hydroxide solution, and the pH of the solution is adjusted to 5-7. EDC is added to activate the carboxyl group in the carboxylated fullerene derivative to form an intermediate active ester. NHS is then added to stabilize the intermediate active ester, thereby reducing the occurrence of side reactions. Finally, amino gellan is added, and an amide reaction is performed to generate gellan fullerene.

[0020] Taking glycine fullerene as an example, the carboxylated fullerene derivative has the following specific synthesis steps: 0.062 g of glycine fullerene was weighed into a 100 mL round-bottom flask, 10 mL of ultrapure water was added to dissolve it, and the mixture was stirred for 1 hour until the solution turned dark brown; 0.046 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.011 g of N-hydroxysuccinimide (NHS) were weighed into the reaction system to activate the carboxyl group for 1 hour, 0.011 mg of amino gellan was weighed into the flask, and the mixture was stirred at room temperature for 40 hours. The solution was transferred to a dialysis bag (3500 Da), dialyzed for 3 days, and freeze-dried to obtain a brown powder after the dialysis was completed.

[0021] A second object of the present invention is to provide an application of the above-mentioned curdlan fullerene derivative.

[0022] The application of the curdlan fullerene derivative provided by the present invention is the application of the curdlan fullerene derivative in the preparation of products for preventing and / or treating liver-related diseases.

[0023] The liver-related diseases mentioned in the present invention include autoimmune hepatitis, viral hepatitis, liver damage, cirrhosis, hepatic fatty degeneration and other liver diseases.

[0024] For example, the product of the present invention may be a medicine or a pharmaceutical preparation.

[0025] In the above applications, when preparing drugs or pharmaceutical preparations, the curdlan fullerene derivative can be used as one of the active ingredients or as the only active ingredient.

[0026] In the above applications, a carrier material may be added when preparing the drug or pharmaceutical preparation.

[0027] Carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to prepare a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc. The formulations can be conventional preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. In order to prepare unit dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparation. The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection; cavity administration, such as rectal and vaginal administration; respiratory administration, such as nasal cavity administration; and mucosal administration.

[0028] A third object of the present invention is to provide a product for preventing and / or treating liver-related diseases.

[0029] The product for preventing and / or treating liver-related diseases provided by the present invention comprises the curdlan fullerene derivative of the present invention as an active ingredient.

[0030] The product may be a drug or a pharmaceutical preparation.

[0031] The product can be prepared into various dosage forms according to conventional methods known to those skilled in the art.

[0032] The curdlan fullerene material of the present invention can be used to treat liver-related diseases such as autoimmune hepatitis, viral hepatitis, liver damage, cirrhosis, hepatic fatty degeneration and other liver diseases through intravenous administration, intraperitoneal administration and the like.

[0033] The curdlan fullerene derivatives described in the present invention treat autoimmune hepatitis, including improving liver tissue inflammatory infiltration, improving liver damage, improving tissue structure (including liver tissue structure and spleen tissue structure), reducing inflammatory factor levels (such as significantly reducing TNF-α and IL-6 levels), reducing alanine aminotransferase and aspartate aminotransferase levels, increasing autoantibodies (antinuclear antibodies, etc.), improving macrophage polarization, improving splenomegaly, etc.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention utilizes curdlan to functionalize fullerene, thereby enhancing the water solubility and biocompatibility of fullerene. By utilizing the macrophage-targeting property of curdlan, the phagocytosis of nanomaterials by liver macrophages can be promoted, thereby enhancing immune regulation and inflammatory resistance.

[0036] (2) The gelatin fullerene material proposed in the present invention can improve liver inflammatory infiltration, tissue damage, increase in autoimmune antibodies, etc. in mice with autoimmune hepatitis, and effectively treat autoimmune hepatitis.

[0037] (3) The curdlan fullerene derivatives provided by the present invention have no obvious toxic side effects on cells and tissues and organs, and have good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1This is the synthesis process of curdlan fullerene;

[0039] Figure 2 This is the H NMR spectrum of curdlan fullerene;

[0040] Figure 3 This is the infrared spectrum of curdlan fullerene;

[0041] Figure 4 Elemental analysis results of curdlan fullerene;

[0042] Figure 5 is the particle size distribution and potential of curdlan fullerene;

[0043] Figure 6 This is an experiment on the antioxidant damage of curdlan fullerene to L02 cells and RAW264.7 cells;

[0044] Figure 7 This is a photo of the mouse liver and spleen;

[0045] Figure 8 is the organ index of mice;

[0046] Figure 9 is the ALT and AST levels in mouse serum;

[0047] Figure 10 The levels of inflammatory factors TNF-α and IL-6 in mice;

[0048] Figure 11 Pathological staining of mouse liver and spleen. DETAILED DESCRIPTION

[0049] The present invention is further described below with reference to specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified.

[0050] Example 1: Synthesis of curdlan fullerene derivatives (flow chart as shown in Figure 1 shown)

[0051] (1) Synthesis of glycine-modified fullerene derivatives:

[0052] Weigh 0.072g of fullerene (C 60) was placed in a 200 mL round-bottom flask, 60 mL of toluene was added to dissolve, and the mixture was stirred for 2 h; 0.75 g of glycine was weighed into a beaker, 8 mL of water was measured and stirred to dissolve until clear, 0.79 g of sodium hydroxide was weighed and stirred to dissolve until clear, and 30 mL of anhydrous ethanol was measured and stirred to dissolve until clear; the clear liquid in the beaker was added dropwise to the fullerene toluene solution, and after reacting for 10 min, five drops of tetrabutylammonium hydroxide solution were added, and the mixture was stirred at 80° C. for 2 days until the purple color in the organic layer disappeared. The anhydrous organic layer was separated and removed, leaving the black aqueous layer, which was diluted with 10 mL of water, and then 50 mL of anhydrous ethanol was added to precipitate. The product was repeatedly precipitated with water and anhydrous ethanol several times, and the solution was transferred to a dialysis bag (3500 Da) and dialyzed for 3 days. After the dialysis was completed, it was freeze-dried to obtain a brown powder, which was the glycine-modified fullerene derivative.

[0053] (2) Preparation method of amino gel polysaccharide:

[0054] The synthesis of amino gellan is divided into two steps. The first step is to modify the C6 position of gellan by azidation to synthesize azidized gellan. The second step is to synthesize amino gellan by reacting azidized gellan with propargyl diglycolamine according to the Click reaction mechanism.

[0055] The first step is to take 0.3g of vacuum-dried curd polysaccharide (purchased from Wako, Japan, 034-09901) into a 100mL round-bottom flask, add 11mL DMF and stir in an 80°C oil bath under nitrogen atmosphere for 30 minutes, weigh 1g NaN3 into the flask, continue stirring for 4 hours to form a uniform white mixed solution, after stirring at 80°C, cool to room temperature, add 1.3g PPh3 and 1.7g CBr4 into the flask in turn, stir at room temperature for 40 hours, the white liquid in the flask first turns into a viscous egg yolk-like liquid, and then turns into a yellow solution, add a little methanol to quench the reaction to produce a white precipitate, add anhydrous methanol, anhydrous ethanol, and deionized water in turn, wash twice and remove impurities, collect the white precipitate and vacuum dry it to obtain a white powder solid.

[0056] The second step is to weigh 0.052g of azido gel polysaccharide into a 50mL round-bottom flask, add 5mL of ultra-dry dimethyl sulfoxide to dissolve it; weigh 0.2g of propargyl diglycolamine (ALK-DGA) into the bottle, add 5mL of ultra-dry dimethyl sulfoxide to dissolve it, and stir for 0.5h. The solution is colorless and transparent; then 0.06g of ascorbic acid, 0.032g of copper bromide and 0.23mL of N,N-diisopropylethylamine are added in sequence. During the reaction, the solution changes from colorless to wine red. The reaction is stirred at room temperature for 3d under a nitrogen atmosphere. The solution is transferred to a dialysis bag (3500Da) and dialyzed for 3 days. After dialysis, it is freeze-dried to obtain a white powder.

[0057] (3) Preparation method of curdlan fullerene derivatives:

[0058] 0.062 g of the glycine-modified fullerene derivative prepared in step 1 was weighed into a 100 mL round-bottom flask, 10 mL of ultrapure water was added to dissolve it, and it was stirred for 1 hour. The solution turned dark brown. 0.046 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.011 g of N-hydroxysuccinimide (NHS) were weighed into the reaction system to activate the carboxyl group for 1 hour. 0.011 mg of the amino gellan prepared in step (2) was weighed into the flask and stirred at room temperature for 40 hours. The solution was transferred to a dialysis bag (3500 Da) and dialyzed for 3 days. After the dialysis, it was freeze-dried to obtain a brown powder, which was the gellan fullerene derivative with a yield of 88.3%.

[0059] Example 2: Curdlan fullerene derivatives (Cur-C 60 )

[0060] (1) H NMR spectroscopy

[0061] The product prepared in Example 1 was subjected to H NMR analysis. Figure 2 As shown in the figure, the chemical shifts at 3.57ppm-3.79ppm are the signal peaks of the hydroxyl group in the curdlan, a new absorption peak appears at 8.46ppm, which is the characteristic absorption peak of the 1,2,3-triazole heterocycle, the chemical shifts at 2.86ppm-3.36ppm are the absorption peaks of the hydrogen atoms on ALK-DGA, and a single peak appears at 2.73ppm, which is the absorption peak of -CH2-. These basically correspond to the product.

[0062] (2) Infrared spectroscopy

[0063] Weigh a small amount of sample glycine fullerene (C 60 -gly), Cur-N3 (azidated curdlan), Cur-NH2 (amino curdlan) and curdlan fullerene (Cur-C 60 ) were placed in 1.5 mL centrifuge tubes, a small amount of dimethyl sulfoxide was added to dissolve it, two drops of solution were dropped onto the gold plate, and then dried in an oven. The transmittance of the product was measured by infrared spectrometer, as shown in FIG. Figure 3 As shown, from top to bottom are glycine fullerene (C 60 -gly), azidated curdlan (Cur-N3), amino-curdlan (Cur-NH2) and curdlan fullerene (Cur-C 60 ) infrared spectrum. At 3000-3500cm -1 The absorption peak of the hydroxyl group on the gel polysaccharide appears between 2900 cm -1The peak that appears is the characteristic peak of the methylene group on the curdlan. Comparing the Cur-N3 spectrum, the absorption peak of the azide group in the Cur-NH2 spectrum (2111.49 cm -1 ) completely disappeared, indicating that Cur-N3 has completely reacted with ALK-DGA after the Click reaction. 60 -gly spectrum, Cur-C 60 In the spectrum, at 2921cm -1 An absorption peak appeared, which was the characteristic peak of the methylene group on the curdlan. From this, it can be judged that Cur-C was successfully prepared. 60 .

[0064] (3) Elemental analysis

[0065] A small amount of curdlan fullerene sample was weighed and placed in a 1.5 mL centrifuge tube. The percentage contents of C, H, and N elements in the product were determined using an elemental analyzer (Thermo Scientific), and the degree of substitution of glycine fullerene in the curdlan fullerene was calculated.

[0066] like Figure 4 As shown, the molecular formula of a single polysaccharide unit amino gellan is C 13 N4O5H 22 , the percentage of C is 49.15, the percentage of N is 6.53, the relative molecular mass of C is 12, and the relative molecular mass of N is 14. The calculation method is as follows:

[0067]

[0068] The calculated DS is 0.6730, which means that on average 67.30 glycine fullerenes are substituted for every 100 polysaccharide units of amino gellan.

[0069] Example 3: Particle size and potential test of curdlan fullerene derivatives

[0070] Weigh 2 mg of curdlan fullerene sample and prepare a 1 mg / mL aqueous solution. Filter with a 0.22 μm filter and ultrasonically vibrate for 5 minutes. Take 1.5 mL and add it to a glass cuvette. Mix well and test. Using ultrapure water as the background, a dynamic light scattering particle size analyzer (Malvern, UK) was used to measure the particle size and potential of the sample. Figure 5 As shown in Figure 1, in aqueous solution (pH = 7.0), the hydrated particle size of curdlan fullerene is 120.2 nm. The Zeta potential of curdlan fullerene is -24.4 mV, and the surface of the nanoparticles is negatively charged, which is beneficial for their application in cells and living organisms.

[0071] Example 4: Antioxidative damage experiment of curdlan fullerene

[0072] The cells that had reached the logarithmic growth phase were cultured at 5×10 4 / mL were seeded in a 96-well plate, with 200μL of culture medium in each well. The plates were incubated in an incubator for 12 hours, the old culture medium was discarded, and each well was washed once with 200μL of PBS buffer, which was discarded. Curdlan fullerene solutions with concentrations of 10μM, 50μM, 100μM, and 200μM were prepared (the solvent was cell culture medium), and 200μL of each sample solution was added to a 96-well plate. The plates were incubated in an incubator for 12 hours, the old culture medium was discarded, and each well was washed once with 200μL of PBS buffer, which was discarded. 200μL of H2O2 solution was added to each 96-well plate, the plates were incubated in an incubator for 1 hour, the H2O2 solution was discarded, and each well was washed once with 200μL of PBS buffer, which was discarded. 10 μL of CCK-8 and 90 μL of colorless DMEM were added to each well and cultured in an incubator for 0.5 h. The absorbance was then read at 450 nm using a microplate reader. The experiment was repeated three times, and the cytotoxicity of the samples was analyzed by plotting using GraphPad Prism8 software.

[0073] like Figure 6 As shown, after L02 cells and RAW264.7 cells were incubated with 1mM H2O2 for 1h, the cell activity was reduced to about 50% of the control group. L02 cells were incubated with curdlan fullerene at different concentrations, and it was found that at a higher concentration of 200μM, curdlan fullerene was able to protect cells against cell death caused by H2O2.

[0074] Example 5: Treatment of autoimmune hepatitis with curdlan fullerene derivatives

[0075] (1) Model establishment

[0076] Thirty-six 7-week-old male Balb / c mice were randomly divided into three groups (6 mice per group): a normal group, a model group, and a curdlan fullerene group (40 mg / kg). The following procedures were performed on the first two days: the normal and model groups were injected with the same dose of normal saline via the tail vein, while the curdlan fullerene group was injected with a curdlan fullerene solution (40 mg / kg, 200 μL, in normal saline). On the final day, the normal group was injected with normal saline via the tail vein, while the remaining groups were injected with concanavalin A (15 mg / kg, 200 μL) via the tail vein. Twelve hours after modeling, the mice were sacrificed for specimen collection and related testing.

[0077] (2) Photography of mouse liver and spleen and organ index

[0078] After sampling, the liver and spleen of the mice were photographed. Figure 7As shown, curdlan fullerene can improve liver damage and spleen enlargement in mice with autoimmune hepatitis. Before killing the mice, the mice were weighed. After killing the mice, the liver and spleen were removed and weighed and recorded. The liver index and spleen index were calculated as follows: liver index (mg×g -1 ) = liver weight (mg) / mouse body weight (g); spleen index (mg×10g -1 ) = spleen weight (mg) / mouse body weight (g) × 10. Figure 8 As shown in the results, fullerene significantly reduced the increase in spleen weight caused by concanavalin and improved spleen enlargement.

[0079] (3) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels

[0080] The changes of ALT and AST in serum were detected by automated blood biochemical analyzer. Figure 9 As shown, curdlan fullerene significantly reduced the elevation of ALT and AST in mice with autoimmune hepatitis and improved liver damage.

[0081] (4) Levels of inflammatory factors TNF-α and IL-6

[0082] The ELISA kit uses a double antibody sandwich method to determine the changes in the levels of TNF-α and IL-6 in mice. The absorbance value of each sample is measured in sequence at a wavelength of 450nm using a microplate reader, and the content of TNF-α and IL-6 in serum samples is calculated using a standard curve. Figure 10 As shown, curdlan fullerene significantly reduced the levels of inflammatory factors in serum and improved inflammation.

[0083] (5) HE staining of liver and spleen

[0084] After sampling, the liver and spleen were fixed with 4% paraformaldehyde and then stained with HE. Figure 11 As shown, the liver cells of mice in the normal group were neatly arranged and clearly visible, radiating outward from the central vein, with no inflammatory cell infiltration. In the model group, the livers of mice had significantly increased lymphocyte infiltration, and a large number of hepatocytes showed focal necrosis. In the curdlan fullerene group, the liver cells showed no obvious degeneration or necrosis, and the liver tissue structure was improved (black arrows indicate necrotic areas). In the normal group, the white and red pulp were evenly distributed, with a clearly visible marginal zone, densely arranged lymphocytes, and intact tissue structure. In the model group, the white pulp of mice had a large number of lymphocytes decreased, the boundary between the red pulp and the marginal zone was unclear, the number of macrophages in the marginal zone decreased, and the overall tissue structure was damaged. In the curdlan fullerene group, the white pulp had a large number of lymphocytes increased, the boundary between the white and red pulp was clear, and the spleen tissue structure was improved.

Claims

1. Use of a curdlan fullerene derivative in the preparation of a drug for preventing and / or treating autoimmune hepatitis; The curdlan fullerene derivative is prepared from curdlan and a fullerene derivative, wherein: The curdlan and the fullerene derivative are covalently linked via amidation; The curdlan is a water-soluble curdlan derivative, which is a water-soluble small molecule with terminal amino groups and alkyne groups modified by a click reaction; The fullerene derivative is a glycine-modified fullerene derivative; The fullerene in the fullerene derivative is C 60 .

2. The use according to claim 1, characterized in that: The molar ratio of the curdlan to the fullerene derivative is 1:(0.5-4), and the fullerene substitution degree of the curdlan fullerene synthesized in different ratios is different, ranging from 0.2 to 0.

9.

3. The use according to claim 1 or 2, characterized in that: The hydrated particle size of the curdlan fullerene derivative is 50-200 nm, and the electric potential is -10 mV to -40 mV.

4. The use according to claim 1 or 2, characterized in that: The preparation method of the curdlan fullerene derivative comprises the following steps: under the activation of EDC and NHS, the amino curdlan and the carboxylated fullerene derivative are subjected to an amide reaction to obtain the curdlan fullerene derivative.

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