Application of amino acid derived carbon dots in preparation of medicine for treating liver injury and / or intestinal barrier injury

Abstract

The invention provides application of amino acid derived carbon dots in preparation of drugs for treating liver injury and / or intestinal barrier injury, and belongs to the technical field of new biomedical materials. Different kinds of amino acids and citric acid monohydrate are used as precursors, a carbonization product is generated through a one-step hydrothermal / solvothermal method, and then separation and purification are performed to prepare the amino acid derived carbon dots, and the prepared amino acid derived carbon dots are small in average particle size, rich in oxygen-containing and nitrogen-containing functional groups on the surface, good in water solubility, free of metal elements, high in biological safety and suitable for industrial production. In addition, good free radical scavenging activity and antioxidant activity as well as good biocompatibility are shown in vitro or in vivo. The compound has the effects of inhibiting liver injury and / or intestinal barrier injury, lowering the inflammation level of nematodes, reducing injury caused by oxidative stress to nematodes, relieving AFB1-induced liver function injury and intestinal barrier injury and regulating intestinal flora, and has a good application prospect in preparation of medicines for treating liver injury and / or intestinal barrier injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of new biomedical materials, and specifically relates to the application of amino acid-derived carbon dots in the preparation of drugs for treating liver damage and / or intestinal barrier damage. Background Art

[0002] Aflatoxin B1 (AFB1) is a contaminant commonly found in food and feed, and its metabolic decomposition produces excessive reactive oxygen species (ROS), which overwhelm the body's antioxidant defense system and lead to oxidative stress, hepatocyte apoptosis, and liver damage. Addressing this oxidative stress is critical to ameliorate AFB1-induced hepatocellular damage and multi-organ toxicity.

[0003] Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor known to regulate various oxidative stress-related diseases. Recent studies have shown that targeting Nrf2 is promising for alleviating AFB1-induced oxidative stress. Normally, Nrf2 binds to kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm, preventing its nuclear translocation and transcriptional activity. However, exposure to oxidants and electrophiles such as AFBO and ROS induces the dissociation of Nrf2 from Keap1, leading to its activation. Activated Nrf2 then translocates to the nucleus, binds to the antioxidant response element (ARE), and promotes the expression of downstream phase II detoxification enzymes and antioxidant proteins. Strategies using antioxidants to remove ROS and activate the antioxidant system, as well as modulating the composition of the intestinal microbiota, are promising for the prevention and treatment of AFB1 toxicity.

[0004] Carbon-based nanomaterials are materials with advantages such as high chemical stability, high catalytic activity, low toxicity and biosafety compared to metal-based nanomaterials, and are expected to play a potential role in the prevention and treatment of inflammatory diseases. Carbon dots (CDs) are a new type of carbon-based nanomaterial with a size less than 10 nm. They have been widely used in the biomedical field due to their inherent activity, small size, good biosafety, easy surface modification and functionalization. CDs can be synthesized from different precursors, such as citric acid, glucose, β-cyclodextrin, L-ascorbic acid, graphene oxide and various biomass materials. Studies have shown that precursors are the key factors affecting the activity of CDs (Gao, Wenhui et al. "Deciphering the catalytic mechanism of superoxide dismutase activation of carbon dot nanozyme." Nature Communications (2023) 14: 160). Amino acids, as the basic building blocks of proteins, play an important role in maintaining nitrogen balance and normal physiological activities in the human body.

[0005] Previous studies have found the application of amino acid-derived carbon dots in ulcerative colitis, but it is still unknown whether they can alleviate aflatoxin B1-induced liver damage or other damage. Summary of the invention

[0006] In view of the above problems, the present invention proposes the use of amino acid-derived carbon dots in the preparation of drugs for treating liver damage and / or intestinal barrier damage.

[0007] Furthermore, the liver damage and / or intestinal barrier damage is induced by aflatoxin B1.

[0008] Furthermore, the amino acid-derived carbon dots include carbon dots derived from any one of histidine, proline, cysteine, tryptophan, aspartic acid, arginine, lysine, methionine or phenylalanine.

[0009] Furthermore, the amino acid-derived carbon dots are arginine-derived carbon dots or proline-derived carbon dots.

[0010] Furthermore, the amino acid-derived carbon dots are prepared by the following method:

[0011] (1) adding amino acids and citric acid monohydrate into a solvent, and heating the mixture to react to obtain an amino acid-derived carbon dot solution;

[0012] (2) The amino acid-derived carbon dots solution is separated and purified to obtain a solid powder, namely the amino acid-derived carbon dots.

[0013] Furthermore, the amino acid and citric acid monohydrate are dissolved in 50-100 mL of a solvent in a mass ratio of 1g:1g to 1g:5g; for example, the mass ratio of the amino acid and citric acid monohydrate can be 1g:g, 1g:2g, 1g:3g, 1g:4g, 1g:5g.

[0014] Furthermore, the solvent includes water and / or an organic solvent.

[0015] Furthermore, the organic solvent includes N,N-dimethylformamide or ethanol.

[0016] Furthermore, the temperature of the heating reaction in step (1) is 160-200° C., and the reaction time is 8-24 h.

[0017] Furthermore, in step (2), separating and purifying the amino acid-derived carbon dot solution comprises:

[0018] The amino acid-derived carbon dot solution is filtered and the filtrate is collected, and then dialyzed using a 500-1000 molecular weight dialysis membrane for 36-72 hours, and vacuum freeze-dried to obtain a solid powder.

[0019] Beneficial effects of the present invention:

[0020] The amino acid-derived carbon dots prepared by the present invention have a small average particle size, rich oxygen-containing and nitrogen-containing functional groups on the surface, good water solubility, no metal elements, no toxicity, high biosafety, and exhibit good free radical scavenging activity and antioxidant activity as well as good biocompatibility both in vitro and in vivo. Compared with natural enzymes, the preparation and purification costs of amino acid-derived carbon dots are low, the chemical properties are stable, and they can work efficiently in harsh environments (strong acids, strong bases) and disease conditions, providing a new idea for the application of nanomaterials in anti-oxidative stress. Based on this, the amino acid-derived carbon dots prepared by the present invention can down-regulate the inflammation level of nematodes, reduce the damage caused by oxidative stress to nematodes, alleviate AFB1-induced damage to mouse liver function and intestinal barrier damage, and regulate intestinal flora, and have good application prospects in the preparation of drugs for treating liver damage and / or intestinal barrier damage.

[0021] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The DPPH radical scavenging ability of the amino acid-derived carbon dots obtained in Example 1 of the present invention is shown;

[0024] Figure 2 The transmission electron microscopy morphology (A) and Fourier transform infrared spectrum (B) of the arginine-derived carbon dots obtained in Example 2 of the present invention are shown;

[0025] Figure 3 The ABTS free radical (ABTS + ) and DPPH free radical (DPPH·) scavenging activity;

[0026] Figure 4The figure shows the biosafety evaluation of the arginine / proline derived carbon dots in Example 3 of the present invention; wherein A is a microscopic photo of nematodes after one week of Arg-CDs feeding; B is the body length statistics of nematodes after one week of Arg-CDs feeding; C is the number of movements of nematodes within 30 seconds after one week of Arg-CDs feeding; D is the hemolysis rate at different concentrations of Arg-CDs;

[0027] Figure 5 The antioxidant capacity evaluation of arginine-derived carbon dots at the nematode level in Example 4 of the present invention is shown; wherein A and B show the effect of Arg-CDs intervention on the ROS fluorescence intensity in nematodes of different treatment groups; C and D are the SOD and MDA contents in nematodes of different treatment groups, respectively;

[0028] Figure 6 The figure shows the situation that the arginine-derived carbon dots alleviate AFB1-induced liver damage in Example 5 of the present invention; wherein A is the experimental scheme of the animal experiment; B is the liver weight of each group of mice; C is the final weight of each group of mice; D is the AST, ALT and ALP levels in the liver; E is the MDA level in the liver; F is the relative expression of inflammatory factors IL-6, IL-1β and TNF-α mRNA in mouse liver tissue; G is the SOD level in serum; H and I are the GSH and CAT contents in the liver, respectively;

[0029] Figure 7 The figure shows the situation that the proline-derived carbon dots alleviate AFB1-induced liver damage in Example 5 of the present invention; wherein A is the experimental scheme of the animal experiment; B is the liver weight of each group of mice; C is the final weight of each group of mice; D and E are the levels of AST and ALT in serum, respectively; F and G are the relative expression levels of inflammatory factors TNF-α mRNA and IL-6 mRNA in mouse liver tissue; H and I are the contents of MDA and GSH in the liver, respectively; J and K are the levels of CAT and SOD in serum, respectively;

[0030] Figure 8 It shows that the arginine-derived carbon dots in Example 6 of the present invention prevent AFB1-induced liver oxidative damage by activating the Nrf2 signaling pathway and the expression of detoxification enzymes mediated by it; wherein A is the relative protein level of CYP450 1A2 in the liver of each group of mice; B is the relative mRNA level of GSTA3 in the liver of each group of mice; C is the relative mRNA level of Nrf2 in the liver of each group of mice; DE is the relative expression of downstream detoxification enzyme genes (GSS, GCLC, GCLM) and antioxidant enzyme genes (SOD, CAT, HO-1, NQO-1) mRNA in the liver of each group of mice, and F and G are the relative expression of Nrf2, Keap1, GCLC, and NQO-1 proteins in the liver of each group of mice;

[0031] Fig. 9 It is shown that the proline-derived carbon dots in Example 6 of the present invention prevent AFB1-induced liver oxidative damage by activating the Nrf2 signaling pathway and the expression of detoxification enzymes mediated by it; wherein AF is the relative expression of Nrf2, Keap1 and antioxidant enzyme genes (SOD, CAT, HO-1, NQO-1) mRNA in the liver of each group of mice; GI is the relative expression of downstream detoxification enzyme genes (GSS, GCLC, GCLM) mRNA in the liver of each group of mice;

[0032] Fig.10 It shows that the arginine-derived carbon dots in Example 7 of the present invention alleviate the changes in intestinal barrier function induced by AFB1; wherein A is a representative small intestine photo of each group of mice and a photo of hematoxylin and eosin staining of ileum tissue; B and C are the ratio of small intestine length and villus height to crypt depth of each group of mice, respectively; D is the relative expression of intestinal tight junction molecules ZO-1, Occludin, Claudin-1 and Muc2 mRNA; EG is the relative expression of Occludin and IL-1β proteins; H and I are the LPS contents in serum and liver, respectively; J is the relative expression of inflammatory factors IL-6, IL-1β and TNF-α mRNA in mouse small intestinal tissue;

[0033] Fig.11 It shows that the proline-derived carbon dots in Example 7 of the present invention alleviate the changes in intestinal barrier function induced by AFB1; wherein A is a representative small intestine photo of each group of mice and a photo of ileum tissue stained with hematoxylin and eosin; B and C are the ratio of the small intestine length and villus height to crypt depth of each group of mice, respectively; DF is the relative expression of inflammatory factors IL-6, IL-1β and TNF-α mRNA in mouse small intestinal tissue; GJ is the relative expression of intestinal tight junction molecules ZO-1, Occludin, Claudin-1 and Muc2 mRNA;

[0034] Fig.12 It shows that the proline-derived carbon dots in Example 8 of the present invention alleviate the changes in the composition of the intestinal microbiota in liver damage induced by AFB1; wherein A and B are studies on α diversity based on Faith_pd and Obs erved_features indices, respectively; C and D are studies on β diversity based on Unweighted_Unifrac and Bray_Curtis indices, respectively; E is the average relative abundance of each group of microbiota at the phylum level in each group of mice in Example 8 of the present invention;

[0035] Fig.13The figure shows the average relative abundance of the microbiota of each group of mice at the genus level and the differential strain analysis in Example 8 of the present invention; wherein A is the average relative abundance of the microbiota of each group of mice at the genus level; BD are the relative abundances of Eubacterium, Akkermansia, and Desulfovibrio in each group of mice, respectively. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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.

[0037] Example 1

[0038] The preparation of arginine-derived carbon dots is as follows:

[0039] (1) Arginine-derived carbon dots (Arg-CDs): 1 g of arginine and 1 g of citric acid monohydrate were weighed and added to 50 mL of double distilled water. The solution was added to a 100 mL polytetrafluoroethylene-lined reactor and heated in an oven at 200 °C for 12 h to obtain a yellow amino acid-derived carbon dot solution.

[0040] (2) Pour the yellow amino acid-derived carbon dot solution into a beaker and filter it with a 0.22 μm filter membrane. Then dialyze it with a 500 molecular weight dialysis membrane for 48 hours, filter the dialyzate with a 0.22 μm filter membrane and pour it into a glass petri dish, put it in a -20°C refrigerator, and freeze-dry it with a vacuum freeze dryer to obtain a solid powder;

[0041] (3) Ultrapure water was added to the solid powder obtained above, and the powder was dissolved by ultrasonication. The insoluble components were removed by filtration using a 0.22 μm water filter. The filtrate obtained was the arginine-derived carbon dots (Arg-CDs).

[0042] According to methods (1)-(3), carbon dots derived from different amino acids were prepared, including proline-derived carbon dots (Pro-CDs), cysteine-derived carbon dots (Cys-CDs), tryptophan-derived carbon dots (Trp-CDs), aspartic acid-derived carbon dots (Asp-CDs), histidine-derived carbon dots (His-CDs), lysine-derived carbon dots (Lys-CDs), methionine-derived carbon dots (Met-CDs) and phenylalanine-derived carbon dots (Phe-CDs).

[0043] Example 2

[0044] The basic physicochemical properties of the amino acid-derived carbon dots prepared in Example 1 were measured.

[0045] (1) DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging activity;

[0046] A 0.1 mM DPPH ethanol solution was prepared, and then 100 μL of amino acid-derived carbon dots (500 μg / mL) was mixed with 1 mL of DPPH solution. After incubation in the dark for 30 min, the absorbance of each solution was measured using a UV spectrophotometer, and the absorption curve was plotted (e.g. Figure 1 As shown in Figure 1, it can be seen that the amino acid-derived carbon dots prepared in Example 1 have the ability to scavenge DPPH free radicals. It can be seen that after the addition of amino acid-derived carbon dots, the characteristic absorption peak of DPPH free radicals in the system at 517 nm is reduced, and the histidine-derived carbon dots (His-CDs) have the best scavenging effect ( Figure 1 ).

[0047] (2) Characterization of morphology and surface functional groups;

[0048] The morphology of arginine-derivatized carbon dots (Arg-CDs) was characterized by JEM-F200 transmission electron microscopy. Fourier transform infrared (FT-IR) spectra were collected using a Bruker Hyperion 2000 spectrometer. Figure 2 As can be seen in Figure A, the prepared Arg-CDs exhibit good monodispersity, have a roughly spherical shape, an average particle size of 4.02 ± 0.82 nm, and a Gaussian distribution in the range of 2-7 nm. In addition, the HRTEM image shows that the lattice fringes of Arg-CDs are clear with an average spacing of 0.32 nm. Figure 2 B is the Fourier transform infrared spectrum of Arg-CDs. Among them, 3206.48cm -1 is the hydroxyl stretching vibration peak, 1657.73cm -1 It is the asymmetric stretching vibration peak of carboxyl C=O.

[0049] (3)ABTS + (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation) and DPPH· free radical scavenging ability assay;

[0050] In order to evaluate the antioxidant capacity of Arg-CDs, the free radical (ABTS + The specific process is as follows:

[0051] Method for determining the free radical scavenging activity of ABTS: First, the ABTS stock solution (14 mM) was reacted with potassium persulfate solution (4.9 mM) under dark conditions for 12 hours to prepare a dark green ABTS solution. The resulting ABTS solution was then diluted with ddH2O until the absorbance at 734 nm reached about 1.0. Subsequently, different concentrations of Arg-CDs solution (ranging from 0 to 500 μg / mL) were added to 1 mL of the ABTS solution. The mixture was incubated in a dark room at 37°C for 6 minutes, and the absorbance value at 734 nm was recorded.

[0052] The DPPH free radical scavenging rate was determined by mixing different concentrations of Arg-CDs aqueous solution with 1 mL of 0.05 mM DDPPH·ethanol solution to prepare Arg-CDs with final concentrations of 0, 5, 10, 50, 100, 200, 300, 400, and 500 μg / mL. After placing in the dark for 20 min, the absorbance of each group of solutions at 517 nm was recorded.

[0053] In the presence of an oxidizing agent, the colorimetric indicator ABTS is oxidized to green ABTS + , while the presence of antioxidants inhibits ABTS + The formation of ABTS + The absorbance at 734 nm can quantify the effect of Arg-CDs on ABTS + cleaning ability.

[0054] Figure 3 A shows that with the increase of Arg-CDs concentration, ABTS + The inhibition rate increased and exceeded 80% at 400 μg / mL. Similarly, Arg-CDs showed effective scavenging of DPPH, a stable free radical with non-bonding electrons (N·) in the molecule. Figure 3 Figure B shows that as the concentration of Arg-CDs increases, the inhibition rate of DPPH· also increases. At 400 μg / mL, the inhibition rate exceeds 60%. From the data, it can be seen that Arg-CDs has excellent antioxidant properties.

[0055] Example 3

[0056] Biosafety Assessment of Amino Acid-Derived Carbon Dots:

[0057] Nematode level: Caenorhabditis elegans (hereinafter referred to as nematodes) was used as experimental animals to explore the effects of feeding Arg-CDs on the body length and movement frequency of nematodes. The experimental groups are as follows:

[0058] The control group (Control) was fed with E. coli OP50, and the experimental group (Arg-CDs) was fed with a mixture of OP50 and 100 μg / mL Arg-CDs. Use a picker to transfer the synchronized L4 nematodes to the corresponding NGM plates. There are at least 3 plates per group, with about 200 nematodes on each plate, and they are inverted and cultured at 20°C. This is recorded as day 0. Transfer the nematodes on each culture plate to a new NGM every one or two days. On the 7th day, randomly pick at least 10 live nematodes in each group using a picker, gently place them on a water agar plate, observe them under a microscope, and measure and record the body length of the nematodes using LAZEZ software. The experiment was repeated three times independently. The body length of the nematodes reflects the growth condition and growth rate of the nematodes. Figure 4 A in the middle is a microscopic photograph of different groups of Caenorhabditis elegans. Figure 4 B and C in the figure respectively counted the body length and movement times of nematodes in each group. The results showed that there was no significant difference in body length and movement times of nematodes fed with mixed diet compared with those of the control group.

[0059] Hemolysis test: The Arg-CDs prepared in Example 1 were subjected to a hemolysis test to investigate its safety. The mice were anesthetized and blood was collected. The blood was centrifuged at 3000rpm for 10 minutes to separate serum, plasma and red blood cells. After the upper serum and plasma were drawn, an appropriate amount of PBS was added to wash the red blood cells. The red blood cells were centrifuged at 3000rpm for 10 minutes and repeated 4-5 times until the supernatant was completely colorless. The red blood cells were configured as a 4% red blood cell solution. A 1mL EP tube was taken, 1mL of 4% red blood cell solution was added, and the red blood cells were centrifuged at 3000rpm for 10 minutes, and the supernatant was discarded. The Arg-CDs were dissolved in PBS and configured into a series of concentrations (100, 2 00, 400, 800ug / ml), take 1m drug PBS solution and add it to the red blood cell sediment, use 1m PBS as negative control, and 1ml H2O as positive control and add it to the red blood cell sediment in step 4; after mixing the above solutions, place them in a 37℃ oven for 3h, then centrifuge at 3000rpm for 10 minutes, observe whether the supernatant has hemolysis, take 100ul of each sample to a 96-well plate to measure the absorbance at 542nm hemolysis rate % = [(test sample-negative) / (positive-negative)] x 100%. The results are as follows Figure 4 As shown in D, the hemolysis rate of Arg-CDs at a concentration of 800ug / ml was less than 1%, proving that it has good biosafety.

[0060] The above experiments demonstrated the safety of Arg-CDs.

[0061] Example 4

[0062] Caenorhabditis elegans was used as the experimental animal, and 0.2 mM Juglone was used to induce the oxidative stress model. After 7 days of culture on NGM plates containing or without Arg-CDs, the synchronized L4 nematodes were collected, and the ROS levels of each group were monitored using ROS detection probes and ROS fluorescence images were obtained using a laser confocal microscope (OLYMPUS FV1000+IX81). Figure 5 As shown in A and B, the mean fluorescence intensity of the experimental group (Juglone+Arg-CDs) decreased compared with the model group (Juglone), indicating that Arg-CDs can alleviate the excessive ROS level in the organism. Commercial analysis kits were used for further analysis to determine the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in C. elegans. SOD is an important antioxidant enzyme that reflects the antioxidant capacity of an organism, while MDA is the final product of lipid peroxidation and can be used as an indicator of oxidative damage. Figure 5 As shown in D, the MDA content in the nematodes in the model group was significantly increased, and the MDA content after Arg-CDs intervention was significantly reduced compared with the model group, indicating that Arg-CDs intervention can effectively alleviate the damage of cells in nematodes in the oxidative stress model. Figure 5 The levels of SOD in nematodes in different treatment groups in C also proved that Arg-CDs intervention can promote the expression of SOD and regulate oxidative stress.

[0063] Example 5

[0064] The Arg-CDs and Pro-CDs in Example 1 were used to treat aflatoxin B1 (AFB1)-induced liver damage in mice. The experimental scheme was as follows: Figure 6 A (Arg-CDs) and Figure 7 As shown in A (Pro-CDs), the mice were randomly divided into four groups (5 mice in each group):

[0065] control group (Control), received 5% DMSO or sterile phosphate-buffered saline (PBS);

[0066] The AFB1 group (AFB1) was orally administered with AFB1 at a dose of 0.75 mg / kg BW, and AFB1 powder was dissolved in 5% DMSO;

[0067] The AFB1 plus Arg-CDs group (AFB1+Arg-CDs) was orally administered with Arg-CDs at a dose of 8 mg / kg BW 8 hours before oral administration of AFB1;

[0068] The AFB1 plus Pro-CDs group (AFB1+Pro-CDs) was orally administered Pro-CDs at a dose of 8 mg / kg BW 8 hours before oral administration of AFB1.

[0069] The body weight and food intake of mice were recorded weekly. After 4 weeks of treatment, mice were weighed, anesthetized and sacrificed, whole blood samples were collected, and then centrifuged (4°C, 2500rpm) for 20 minutes to obtain serum, which was stored at -80°C until analysis. Liver tissue was removed and weighed, and small intestine and colon tissue were collected and measured in length. Liver and small intestine samples were stored at -80°C for subsequent analysis.

[0070] like Figure 6 As shown in B and C, after 4 weeks of experimental period, Arg-CDs intervention alleviated the liver weight of mice induced by AFB1 ( Figure 6 B) and final body weight ( Figure 6 C) decrease.

[0071] Serum biochemical factors indicative of liver damage: alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were quantified using commercial test kits (Nanjing Jiancheng Bioengineering Institute, China). Figure 6 Arg-CDs alleviated the AFB1-induced increase in ALT, AST, and ALP levels as shown in D. Superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) are indicators of the capacity of the human enzymatic antioxidant defense system.

[0072] The activities of MDA and GSH in liver homogenate supernatant and SOD and CAT in serum were determined according to the instructions of the corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute, China). Figure 6 E) while inhibiting SOD ( Figure 6 GSH Figure 6 H) and CAT ( Figure 6 In contrast, Arg-CDs can alleviate the inhibitory effect of AFB1 on antioxidant enzyme activity and reduce MDA levels. Real-time fluorescence quantitative PCR was used to detect the relative expression of inflammatory factors in the liver tissues of each group of mice. The results showed that Arg-CDs can downregulate the expression of proinflammatory factors Il-6, IL-1β, TNF-α ( Figure 6 F) mRNA expression level.

[0073] like Figure 7 As shown in B and C, after 4 weeks of experimental period, Pro-CDs intervention alleviated AFB1-induced liver weight loss in mice ( Figure 7 B) and final body weight ( Figure 7 C) is reduced. Figure 7As shown in D and E, Pro-CDs alleviated the increase of AST and ALT levels induced by AFB1. Real-time fluorescence quantitative PCR was used to detect the relative expression of inflammatory factors in the liver tissues of mice in each group. The results showed that Arg-CDs could downregulate the expression of pro-inflammatory factors TNF-α, Il-6 ( Figure 7 Pro-CDs inhibit the production of MDA ( Figure 7 H) and promote GSH ( Figure 7 Medium I), CAT( Figure 7 J) and SOD ( Figure 7 The K) activity can alleviate AFB1-induced oxidative damage in hepatocytes.

[0074] These results indicate that Arg-CDs and Pro-CDs can alleviate AFB1-induced liver injury by regulating oxidative stress and inflammatory responses.

[0075] Example 6

[0076] Previous studies have found that Arg-CDs and Pro-CDs can reduce AFB1-induced liver oxidative damage by enhancing the activity of antioxidant enzymes. This example uses the Arg-CDs and Pro-CDs in Example 1 to treat aflatoxin B1 (AFB1)-induced liver damage in mice. In order to understand how to reduce liver oxidative damage, the potential mechanism is further explored, and the experimental process and results are explained using Arg-CDs and Pro-CDs as examples.

[0077] In the metabolism of AFB1, the phase I metabolic enzyme CYP450s is responsible for converting AFB1 into its harmful metabolites. The relative expression level of the protein of the phase I metabolic enzyme CYP450 1A2 was detected by Western Blot (WB) in each group of mice. Figure 8 As shown in Figure A, the expression of CYP450 1A2 protein in the liver of mice was significantly increased after AFB1 exposure, while Arg-CDs showed a trend of downregulating the expression of this protein. In phase II metabolism, detoxification enzymes (such as the glutathione S-transferase (GST) family, such as GSTA3) play a vital role in alleviating the toxicity of AFB1. Figure 8 As shown in B, Arg-CDs significantly reversed the decrease in GSTA3 expression caused by AFB1.

[0078] The nuclear factor erythroid 2-related factor 2 (Nrf2) / Kelch-like ECH-associated protein 1 (Keap1) signaling pathway has a clear protective effect in AFB1-induced hepatotoxicity. Real-time fluorescence quantitative PCR and protein immunoblotting (Western Blot, WB) were used to detect the relative expression levels of related signaling molecules in the Nrf2 / Keap1 signaling pathway in the liver of each group of mice. Figure 8 As shown in Figure CG, after Arg-CDs intervention, the dissociation of Keap1 and Nrf2 was promoted, which was manifested by a significant increase in Nrf2 gene expression, a significant decrease in Keap1 protein expression, and a significant upregulation of downstream detoxification enzyme genes (GSS, GCLC, GCLM, Figure 8 D) and antioxidant enzyme genes (SOD, CAT, HO-1, NQO-1, Figure 8 Western blot results confirmed these findings ( Figure 8 At the same time, Pro-CDs intervention also promoted the dissociation of Keap1 and Nrf2, which was manifested by a significant increase in Nrf2 gene expression ( Fig. 9 Middle A), Keap1 protein expression was significantly reduced ( Fig. 9 In the middle B), antioxidant enzyme genes (SOD, CAT, HO-1, NQO-1, Fig. 9 CF) and downstream detoxification enzyme genes (GSS, GCLC, GCLM, Fig. 9 These results suggest that Arg-CDs and Pro-CDs can alleviate liver oxidative damage by activating the Nrf2 / Keap1-mediated oxidative stress pathway.

[0079] Example 7

[0080] The Arg-CDs prepared in Example 1 were used to alleviate the intestinal barrier damage in mice with liver damage induced by aflatoxin B1 (AFB1). Fig.10 As shown in B, Arg-CDs intervention alleviated the shortening of small intestine length in mice with liver injury. H&E stained ileum histopathological analysis showed that Arg-CDs intervention alleviated the AFB1-induced decrease in the ratio of villus height to crypt depth and the destruction of the integrity of small intestinal villi ( Fig.10 Real-time fluorescence quantitative PCR and protein immunoblotting (Western Blot, WB) were used to detect the relative expression levels of intestinal tight junction molecules and inflammatory factors in each group of mice. Fig.10In the DG, Arg-CDs alleviated the decrease in the relative expression levels of intestinal tight junction molecules (ZO-1, Occludin, Claudin-1, and Muc2) and the increase in the level of the inflammatory factor IL-1β caused by AFB1 exposure, indicating the beneficial effects of Arg-CDs in improving intestinal barrier damage in AFB1-exposed mice.

[0081] Lipopolysaccharide (LPS) levels in serum and liver were measured by enzyme-linked immunosorbent assay (ELISA). LPS is a unique component of the outer membrane of Gram-negative bacteria and can serve as a virulence factor and protective antigen. Under various pathological conditions, bacteria can break through the intestinal mucosal barrier, enter the blood, reach the liver through the "gut-liver axis" pathway, trigger hepatocyte immune responses, and cause liver disease through immune-inflammatory mechanisms. AFB1 exposure can increase LPS levels in the blood and liver, while Arg-CDs intervention can reverse this phenomenon ( Fig.10 H, I). Real-time fluorescence quantitative PCR was used to detect the relative expression of inflammatory factors in the colon tissue of each group of mice. The results showed that Arg-CDs intervention significantly reduced the expression of inflammatory cytokines IL-6, IL-1β and TNF-α in the ileum ( Fig.10 Based on these findings, the amino acid-derived carbon dots prepared in the present invention may prevent toxic substances from leaking from the intestine by regulating the intestinal barrier function, thereby alleviating oxidative stress and inflammatory responses in the body and reducing organ damage, including liver damage.

[0082] The Pro-CDs prepared in Example 1 were used to alleviate the intestinal barrier damage in mice with liver damage induced by aflatoxin B1 (AFB1). Fig.11 As shown in B, Pro-CDs intervention alleviated the shortening of small intestine length in mice with liver injury. H&E stained ileum histopathological analysis showed that Pro-CDs intervention alleviated the AFB1-induced decrease in the ratio of villus height to crypt depth and the destruction of the integrity of small intestinal villi ( Fig.11 Real-time fluorescence quantitative PCR was used to detect the relative expression of intestinal tight junction molecules and inflammatory factors in each group of mice. Fig.11 In the middle DF, Pro-CDs alleviated the increase in the relative expression levels of inflammatory factors IL-6, IL-1β, and TNF-α and intestinal tight junction molecules (ZO-1, Claudin-1, Occludin, and Muc2) caused by AFB1 exposure ( Fig.11 The decreased relative expression level of Pro-CDs in GJ (Fig.

[0083] Example 8

[0084] This example investigates the effects of the amino acid-derived carbon dots prepared in Example 1 on the alleviation of aflatoxin B1 (AFB1)-induced liver damage in mice, explores the changes in the intestinal microbiota of mice, and uses Pro-CDs as an example to illustrate the experimental process.

[0085] The Pro-CDs in Example 1 were used to alleviate liver damage in mice induced by aflatoxin B1 (AFB1). After 28 days, the feces of mice in each group were collected to explore the changes in the intestinal microbiota of mice. Fig.12 As shown in A and B, the alpha diversity index (Faith_pd and Observed_features) showed that the diversity of intestinal flora in the AFB1 group was reduced compared with that in the control group. In addition, unifrac-based unweighted principal coordinate analysis (PCoA) and Bray_Curtis analysis revealed obvious clustering of the microbiota composition in each treatment group ( Fig.12 These results suggest that Pro-CDs intervention regulates AFB1-induced changes in the intestinal microbiota of mice.

[0086] At the phylum level, Firmicutes and Bacteroidetes were the dominant phyla in the fecal microbiota ( Fig.12 At the genus level, compared with the AFB1 group, the relative abundance of Eubacterium and Akkermansia in the intestines of the Pro-CDs group mice increased significantly, while the relative abundance of Desulfovibrio decreased significantly ( Fig.13 AD). Studies have shown that Eubacterium can produce short-chain fatty acids to provide energy sources for intestinal mucosal cells, promote intestinal epithelial cell proliferation, maintain intestinal barrier, and resist inflammation. Akkermansia can significantly improve liver diseases by regulating inflammatory factors, reducing oxidative stress, enhancing immune function, and remodeling intestinal flora through the intestinal-liver axis. Desulfovibrio can increase intestinal permeability and inflammatory response, leading to gastrointestinal diseases.

[0087] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of amino acid-derived carbon dots in the preparation of drugs for the treatment of liver injury and / or intestinal barrier injury.

2. The use according to claim 1, characterized in that: The liver damage and / or intestinal barrier damage is induced by aflatoxin B1.

3. The use according to claim 1, characterized in that: The amino acid-derived carbon dots include carbon dots derived from any one of histidine, proline, cysteine, tryptophan, aspartic acid, arginine, lysine, methionine or phenylalanine.

4. The use according to claim 1, characterized in that: The amino acid-derived carbon dots are arginine-derived carbon dots or proline-derived carbon dots.

5. The use according to any one of claims 1 to 4, characterized in that: The amino acid-derived carbon dots were prepared by the following method: (1) adding amino acids and citric acid monohydrate into a solvent, and heating the mixture to react to obtain an amino acid-derived carbon dot solution; (2) The amino acid-derived carbon dots solution is separated and purified to obtain a solid powder, namely the amino acid-derived carbon dots.

6. The use according to claim 5, characterized in that The amino acid and citric acid monohydrate are dissolved in 50-100 mL of a solvent in a mass ratio of 1g:1g to 1g:5g.

7. The use according to claim 5, characterized in that: in, The solvent includes water and / or an organic solvent.

8. The use according to claim 7, characterized in that: The organic solvent includes N,N-dimethylformamide or ethanol.

9. The use according to claim 5, characterized in that The temperature of the heating reaction in step (1) is 160-200° C., and the reaction time is 8-24 hours.

10. The use according to claim 5, characterized in that In step (2), separating and purifying the amino acid-derived carbon dot solution comprises: The amino acid-derived carbon dot solution is filtered and the filtrate is collected, and then dialyzed using a 500-1000 molecular weight dialysis membrane for 36-72 hours, and vacuum freeze-dried to obtain a solid powder.

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