Application of cyanidin-3-O-glucoside as medicine for treating / preventing liver injury caused by aflatoxin

Abstract

The invention discloses an application of cyanidin-3-O-glucoside in preparation of a medicine for improving pyroptosis of liver cells caused by aflatoxin B1 (aflatoxin B1). According to the application disclosed by the invention, the C3G is found to have a very good relieving and improving effect on the pyroptosis of liver cells caused by AFB1 (Adenosine Factor B1), the effect of the C3G with a medium dose (3mg / kg) is optimal, and the C3G with the medium dose can be used for obviously inhibiting the expression level of liver pyroptosis pathway related factors and the expression of inflammatory factors such as TLR4, NLRP3 and Caspase-1. The cyanidin-3-O-glucoside is extracted from natural plants in nature, has natural activity and small toxic and side effects, has good improvement and alleviation effects on the pyroptosis of liver cells caused by AFB1, can be used as a medicine for treating the pyroptosis of liver cells caused by AFB1, and can be used as a medicine for treating the pyroptosis of liver cells caused by AFB1. And a novel detoxification scheme is provided for clinical treatment of liver injury caused by AFB1.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more particularly to the use of cyanidin-3-O-glucoside as a drug for treating / preventing liver damage caused by aflatoxin. Background Art

[0002] Aflatoxins (AFs) are bifuranoid toxins produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. There are approximately 20 derivatives, designated as B1, B2, G1, G2, M1, M2, GM, P1, Q1, and AFB1. Aflatoxin 1 is the most toxic and carcinogenic. Aflatoxins primarily contaminate grains, oilseeds, and their products, but can also contaminate various plant and animal foods. Aflatoxins can easily parasitize and produce toxins in cereals and oilseed seeds with high moisture content (less than 12% moisture cannot reproduce), as well as in their processed byproducts. This causes mold and spoilage. People can ingest these foods or their processed byproducts and absorb the toxins through the digestive tract, leading to poisoning. Aflatoxins are potent hepatotoxins with carcinogenic and mutagenic properties. Excessive intake of aflatoxins can damage and kill liver cells, severely impairing liver function. Long-term aflatoxin exposure can also lead to diseases such as cirrhosis and liver cancer.

[0003] Among them, hepatocyte pyroptosis, as a highly inflammatory form of programmed cell death, is the main inducing factor and an important factor in the further development of AFB1-induced liver disease. As proposed in "Research Progress on the Pathogenic Mechanism and Prevention of Aflatoxin B1" (China Animal Husbandry and Veterinary Medicine 2022, 49(2)), pyroptosis is a recently discovered type of programmed inflammatory necrosis characterized by plasma membrane rupture and membrane perforation, which plays a vital role in the body's physiological process of resisting the invasion of exogenous pathogens and detecting internal risk factors. Pyroptosis can be induced by the classical pyroptosis pathway of inflammasomes activating Caspase-1 and the non-classical pyroptosis pathway of cytoplasmic LPS activating Caspase-4 / 5 / 11. After AFB1 invades the body, it can increase the ROS content in the body, causing it to act on the NLRP3 inflammasome and induce the classical pyroptosis pathway of hepatocytes. Hepatocyte pyroptosis will release a large amount of proinflammatory factors, activate the immune cascade reaction, and cause inflammatory damage to hepatocytes. The NLRP3 inflammasome is a multiprotein polymer with pro-inflammatory properties. Its structure contains a caspase adaptor protein, ASC, which activates the intracellular cysteine protease Caspase-1 and promotes the maturation and secretion of 1L-1β pro-1L-1β (Pro-1L-1β) and 1L-18 pro-1L-18 (Pro-1L-18). Activated Caspase-1 activates the cytoplasmic GSDMD protein, cleaving it into GSDMD-N and GSDMD-C. GSDMD-N can penetrate the cell membrane of animal models and form pores, leading to cell pyroptosis.

[0004] Anthocyanins, a class of polyphenolic flavonoids, are widely found in nature as natural pigments, particularly in colorful plants and fruits such as purple sweet potatoes, strawberries, and cherries. Anthocyanins extracted from foods or plants often contain different anthocyanogenins, such as cyanidin, pelargonidin, petunidin, and malvidin. Glycosides can also be classified into mono-, disaccharide-, and tri-glucosides. These monomeric anthocyanins exhibit distinct physiological activities. Anthocyanins have numerous therapeutic effects, including antioxidant, anti-atherosclerotic, anti-insulin resistance, and lipid regulation. Cyanidin-3-O-glucoside (C3G) is the primary active ingredient.

[0005] Cyanidin-3-O-glucoside originates from plants and other natural sources, has few toxic side effects, is abundant in sources, and is inexpensive. However, there are currently no reports on the effect of cyanidin-3-O-glucoside on improving AFB1-induced liver cell pyroptosis. Summary of the Invention

[0006] Based on this, it is necessary to provide a method for treating / preventing liver damage caused by aflatoxin by providing a cyanidin-3-O-glucoside as a drug for the treatment / prevention of liver damage caused by aflatoxin B1, and to provide an efficient and natural detoxification solution for the treatment / prevention of liver cell pyroptosis caused by aflatoxin B1.

[0007] In order to solve the above technical problems, the present invention provides a use of cyanidin-3-O-glucoside as a drug for treating / preventing liver damage caused by aflatoxin, using the following technical solution:

[0008] In a first aspect, the present invention provides a use of cyanidin-3-O-glucoside as a drug for treating / preventing liver damage caused by aflatoxin.

[0009] Furthermore, the aflatoxin is aflatoxin AFB1.

[0010] Furthermore, in animal experiments, the effective dosage of cyanidin-3-O-glucoside was 1.5 to 4.5 mg / kg.

[0011] Furthermore, in animal experiments, the effective dose of cyanidin-3-O-glucoside was 3 mg / kg.

[0012] Furthermore, the improvement was achieved by reducing the pathway proteins of liver cell pyroptosis.

[0013] Furthermore, the pathway protein of liver cell pyroptosis is any one or more of TLR4, NLRP3, Caspase-1, GSDMD, IL-1β and IL-18.

[0014] The second aspect of the present invention provides the use of cyanidin-3-O-glucoside in the preparation of a preparation for reducing any of the above-mentioned liver cell pyroptosis pathway proteins.

[0015] A third aspect of the present invention provides a drug for treating / preventing liver damage caused by aflatoxin, comprising cyanidin-3-O-glucoside.

[0016] Furthermore, the above-mentioned drug for treating / preventing liver damage caused by aflatoxin also includes a pharmaceutically acceptable carrier and is prepared into different dosage forms.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention establishes an AFB1-intoxicated model in normal mice by gavage, and evaluates the effect of AFB1 on inducing pyroptosis of liver cells in mice by detecting changes in biochemical indicators in serum and liver, liver tissue lesions, liver cell pyroptosis pathway proteins, and serum characteristic enzyme activities. The results showed that the LPS dose in the serum and liver of mice in the AFB1 group increased significantly, activated the liver cell pyroptosis signaling pathway, and activated a large number of proinflammatory factors; at the same time, the activity of characteristic enzymes (GOT, GPT, and LDH) in the serum of mice was significantly increased after AFB1 exposure. The present invention found that C3G has a good alleviating and improving effect on liver cell pyroptosis caused by AFB1, among which the medium dose of C3G (3 mg / kg) has the best effect. The medium dose of C3G can significantly reduce the LPS concentration in serum and liver, inhibit the liver cell pyroptosis pathway, reduce the expression levels of liver pyroptosis pathway-related factors (TLR4, NLRP3, and Caspase-1), inhibit a large number of proinflammatory factors, and significantly reduce the release of characteristic enzymes (GPT, GOT, and LDH) from liver cells to the blood.

[0019] The cyanidin-3-O-glucoside in the present invention is extracted from natural plants in nature, has natural activity, and has few toxic and side effects. Experiments in the present invention have shown that cyanidin-3-O-glucoside has a good improvement and alleviation effect on liver cell pyroptosis caused by AFB1, and can be used as a drug for treating liver cell pyroptosis caused by AFB1. It also provides a new detoxification solution for the clinical treatment of liver damage caused by AFB1. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction will be given below to 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 application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0021] Figure 1 The weight change trends of mice in each group in Example 1 of the present invention are shown in Table 1. *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 2 are the organ indexes of mice in each group in Example 1 of the present invention, *P<0.05, **P<0.01,

[0023] ***P < 0.001;

[0024] Figure 3 The LPS content in the liver of each group of mice in Example 1 of the present invention, *P<0.05, **P<0.01, ***P<0.001;

[0025] Figure 4 The LPS levels in the serum of mice in each group in Example 1 of the present invention are *P<0.05, **P<0.01, ***P<0.001;

[0026] Figure 5 The results of pathological analysis of liver tissues in the control group, AFB1 group, and medium-dose C3G group in Example 2 of the present invention are shown;

[0027] Figure 6 The expression levels of hepatocyte pyroptosis pathway proteins in the control group, AFB1 group, and medium-dose C3G group in Example 3 of the present invention, *P<0.05, **P<0.01, ***P<0.001;

[0028] Figure 7 The serum characteristic enzyme GOT activity levels of the control group, AFB1 group and medium-dose C3G group in Example 3 of the present invention, *P<0.05, **P<0.01, ***P<0.001;

[0029] Figure 8 The serum characteristic enzyme GPT activity levels of the control group, AFB1 group and medium-dose C3G group in Example 3 of the present invention, *P<0.05, **P<0.01, ***P<0.001;

[0030] Figure 9 These are the serum characteristic enzyme LDH activity levels of the control group, AFB1 group, and medium-dose C3G group in Example 3 of the present invention, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions 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 embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0032] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0033] 1. Materials and Reagents

[0034]

[0035] 2. Experimental Animals

[0036] Thirty Balb / C male mice aged 3 to 5 weeks, weighing approximately 20 g, were purchased from the Guangdong Medical Experimental Animal Center. The median lethal dose (LD50) of AFB1 administered orally to mice was approximately 4.8 mg / kg.

[0037] Example 1

[0038] 1. Prepare AFB1 working solution

[0039] Dissolve 10 mg of AFB1 standard powder in 10 mL of acetonitrile to prepare a 1 mg / mL AFB1 stock solution (store at -20°C). Dilute 1 mL of the AFB1 stock solution to 50 mL with 40% ethanol (anhydrous ethanol:water = 4:6) to prepare a 20 μg / mL AFB1 stock solution (store at -20°C). Dilute 0.625 mL of the AFB1 stock solution to 5 mL with DMSO to prepare a 2.5 μg / mL AFB1 working solution for oral gavage experiments in mice (prepare immediately before use).

[0040] 2. Grouping of experimental animals

[0041] Thirty male Balb / c mice were randomly divided into five groups, with six mice in each group.

[0042] Control group (CON): mice were given normal saline by intra-gastric gavage (ig) every day for 28 days;

[0043] AFB1 group (AFB1): mice were orally gavaged with normal saline every day for 21 days, followed by orally gavage with 25 μg / kg.bw AFB1 solution (1 / 192 LD50 ig) for 7 days;

[0044] Low-dose C3G group (AFB1+Low C3G): mice were orally gavaged with C3G solution every day for 21 days, with a daily intake of C3G of 1.5 mg / kg, followed by oral gavage of 25 μg / kg AFB1 (1 / 192 LD50 ig) for 7 days;

[0045] Medium-dose C3G group (AFB1+Medium C3G): mice were orally gavaged with C3G solution every day for 21 days, with a daily intake of C3G of 3 mg / kg, followed by oral gavage of 25 μg / kg AFB1 (1 / 192 LD50 ig) for 7 days;

[0046] High-dose C3G group (AFB1+High C4G): Mice were orally gavaged with C3G solution every day for 21 days, with a daily intake of C3G of 4.5 mg / kg, followed by oral gavage of 25 μg / kg AFB1 (1 / 192 LD50 ig) for 7 days.

[0047] 3. Determination of Mouse Weight Growth Rate and Organ Index

[0048] The body weight of the mice was recorded daily, and the weight gain rate of each group was calculated. On day 29, the mice were sacrificed by cardiac venous exsanguination. The livers were collected, washed with phosphate buffered saline (PBS), dried, and weighed. The liver organ index of each group was calculated.

[0049] 4. Evaluate the effects of low, medium, and high doses of C3G on AFB1-exposed mice

[0050] 4.1 Effects of different doses of C3G on body weight changes in AFB1-exposed mice

[0051] Table 1 shows the relative changes in body weight of mice in each group. Figure 1 As shown in the results, compared with the control group, the body weight of mice in the AFB1-exposed group decreased by 27.29% (p<0.001), while the body weight of mice in the low, medium, and high doses of C3G groups decreased by 22%, 14.19%, and 20.79%, respectively. This indicates that AFB1 exposure significantly affects the growth of mice, and that the three doses of C3G alleviated the weight loss of mice. Among them, the medium dose of C3G had the most significant alleviating effect (p<0.01).

[0052] Table 1: Relative changes in body weight of mice in each group (%)

[0053]

[0054] 4.2 Effects of different doses of C3G on organ indices in AFB1-exposed mice

[0055] The damage of C3G exposure to AFB1 in mice was evaluated by measuring the liver weight of each group of mice. Organ index calculation: organ index = organ mass / total mouse weight * 100%. The mouse liver was weighed using a 100% scale. The results are shown in Table 2 and Figure 2 As shown in the results, compared with the control group, the liver organ index of mice in the AFB1 group was significantly increased (p<0.01). Compared with the AFB1 group, the liver weight of mice in the medium-dose C3G group decreased most significantly (p<0.05), while there was no significant difference in liver weight between the low-dose and high-dose C3G groups (p>0.05), indicating that C3G at a concentration of 3 mg / kg can significantly alleviate AFB1-induced liver damage.

[0056] Table 2: Organ index of liver of mice in each group (%)

[0057]

[0058] 4.3 Effects of different doses of C3G on LPS concentrations in the liver and serum of AFB1-exposed mice

[0059] The LPS content in the liver and serum of each group of mice was detected by ELISA kit. The experimental results are as follows: Figure 3 As shown in the results, compared with the control group, the LPS concentration in the liver of the AFB1-treated group increased significantly by 231.94% (p<0.001), while the LPS concentrations of the mice in the low, medium and high dose C3G groups decreased by 17.34%, 54.34% and 43.41% respectively compared with the AFB1 group, and the LPS concentrations in the liver of the mice in the medium dose C3G group decreased most significantly (p<0.001), indicating that medium concentration C3G can effectively reduce the LPS concentration in the liver. Figure 4 As shown, compared with the control group, the LPS dose in the serum of the AFB1-treated group was significantly increased by 291.42% (p<0.001), while relative to the AFB1 group, the LPS levels of mice in the low, medium and high doses of C3G groups were reduced by 26.62%, 60.78% and 45.68%, respectively, and the LPS concentration in the serum of mice in the medium dose of C3G group decreased most significantly (p<0.001).

[0060] Example 2 Histopathological Detection

[0061] 1. Collect liver samples, wash with PBS, and fix with 4% paraformaldehyde at 4°C for 24 hours. Embed in paraffin and slice at 5 μm thickness. Dewax the liver sections in xylene and rehydrate them in a gradient of anhydrous ethanol, 95% ethanol, 85% ethanol, 75% ethanol, and 50% ethanol.

[0062] 2. Stain the nuclei and cytoplasm of liver sections using the recommended staining protocol for the hematoxylin & eosin kit. After staining, dehydrate the sections using a gradient of 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol. Transparent the sections with fresh xylene and mount them with neutral gum. Observe the liver histopathological changes using an inverted fluorescence microscope.

[0063] 3. HE staining test results

[0064] like Figure 5 As shown, pathological analysis of liver tissue also showed that the livers of mice in the AFB1 group showed extensive inflammatory infiltration, hepatocyte hypertrophy, and disappearance of cell membrane vacuolation, while the liver symptoms of mice in the medium-dose C3G group were significantly less.

[0065] Example 3 q-PCR method to determine the transcription level of liver protein gene

[0066] 1. Total RNA from the liver was extracted using the TransZol Up method. The specific procedure was as follows: 0.1 g of liver tissue sample was ground into a powder using liquid nitrogen and transferred to a centrifuge tube. 1 mL of TransZol Up and 0.2 mL of RNA extraction reagent were added, mixed, and shaken at room temperature for 5 minutes. The colorless aqueous phase was obtained by centrifugation (10,000 rpm, 15 minutes, 4°C). This was transferred to a new centrifuge tube, 0.5 mL of isopropanol was added, mixed, and incubated at room temperature for 10 minutes. The sample was centrifuged (10,000 rpm, 10 minutes, 4°C), the supernatant discarded, and 1 mL of 75% ethanol (prepared with DEPC water) was added. The sample was vortexed vigorously to remove salt from the RNA precipitate. The sample was centrifuged (7500 rpm, 5 minutes, 4°C), the supernatant discarded, the precipitate air-dried at room temperature, and 75 μL of RNA dissolution buffer was added. The sample was incubated in a 60°C oven for 10 minutes. The total RNA concentration and purity of the samples were measured using a micro-spectrophotometer and stored at -80°C until use.

[0067] 2. Take an equal amount of RNA from each group of liver samples and use One-step gDNA Removal and cDNA Synthesis SuperMix Kit was used to reverse transcribe RNA into cDNA. Real-time quantitative PCR (q-PCR) was performed on the reverse-transcribed cDNA using the Green qPCR SuperMix kit. Target genes were amplified in a 20 μL reaction mixture, and the assay was repeated three times for each target gene in all samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene, and 2 -ΔΔCT The relative transcription levels of pyroptosis pathway proteins (TLR4, NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18) in liver cells were analyzed using a PCR amplification assay. The CT values of gene transcriptional expression were calculated using ABIQuantity Studio. The forward and reverse primer sequences for each target gene are shown in Table 3.

[0068] Table 3: Sequence list of forward and reverse primers for each target gene

[0069]

[0070] 3. Effects of medium-dose C3G on AFB1-exposed mice

[0071] 3.1 Effects of medium-dose C3G on hepatocyte pyroptosis in AFB1-exposed mice

[0072] The effect of medium-dose C3G on hepatocyte pyroptosis in AFB1-exposed mice was evaluated by analyzing the gene transcription levels of hepatocyte pyroptosis pathway proteins (TLR4, NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18). The gene transcription levels of six proteins related to the hepatic pyroptosis pathway were determined by q-PCR. Figure 6 As shown, the relative mRNA expression levels of TLR4, NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18 in the liver of mice in the AFB1 group were significantly increased (p<0.01), while the relative mRNA expression levels of TLR4, NLRP3, Caspase-1, IL-1β, and IL-18 in the medium-dose C3G group were significantly decreased compared with the AFB1 group (p<0.05). The relative mRNA expression level of GSDMD was not significantly different from that in the control group (p>0.05). These results suggest that medium-dose C3G has a significant alleviating effect on AFB1-induced hepatocyte pyroptosis.

[0073] 3.2 Effects of medium-dose C3G on serum physiological and biochemical parameters in AFB1-exposed mice

[0074] The levels of serum characteristic enzymes (GPT, GOT, LDH) in the control group, AFB1 group and medium-dose C3G group were measured. Figures 7-9 As shown in the data, after AFB1 exposure, the activities of GPT, GOT and LDH in the serum of mice were significantly increased, which were 265.69% (p<0.05), 331.59% (p<0.05) and 68.82% (p<0.001) higher than those in the control group, respectively. This proves that the liver damage is severe, which may be due to the activation of the liver cell pyroptosis signaling pathway, resulting in rupture of the plasma membrane and the influx of characteristic enzymes from the liver cells into the blood. The intake of medium concentration of C3G significantly reversed this situation, which was reduced by 72.64% (p>0.05), 44.69% (p<0.05) and 29.94% (p<0.001) respectively compared with the AFB1 group, further indicating that medium dose of C3G can effectively alleviate the symptoms of AFB1-induced liver cell pyroptosis.

[0075] Obviously, the embodiments described above are only some of the embodiments of the present application, rather than all of the embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions described in the aforementioned specific embodiments, or to make equivalent replacements for some of the technical features therein. Any equivalent structure made using the contents of the present application specification and the accompanying drawings, directly or indirectly used in other related technical fields, is also within the scope of patent protection of the present application.

Claims

1. Use of cyanidin-3-O-glucoside as a drug for the treatment / prevention of aflatoxin-induced liver damage.

2. The use of cyanidin-3-O-glucoside according to claim 1 as a drug for treating / preventing liver damage caused by aflatoxin, characterized in that: The aflatoxin is aflatoxin AFB1.

3. Use of cyanidin-3-O-glucoside according to claim 2 as a drug for treating / preventing liver damage caused by aflatoxin, characterized in that: In animal experiments, the effective dosage of cyanidin-3-O-glucoside is 1.5 to 4.5 mg / kg.

4. Use of cyanidin-3-O-glucoside according to claim 3 as a drug for treating / preventing liver damage caused by aflatoxin, characterized in that: In animal experiments, the effective dose of the cyanidin-3-O-glucoside is 3 mg / kg.

5. Use of cyanidin-3-O-glucoside according to claim 1 as a drug for treating / preventing liver damage caused by aflatoxin, characterized in that: The improvement was achieved by reducing the expression level of pyroptosis pathway proteins in liver cells.

6. Use of cyanidin-3-O-glucoside according to claim 5 as a drug for treating / preventing liver damage caused by aflatoxin, characterized in that: The liver cell pyroptosis pathway protein is any one or more of TLR4, NLRP3, Caspase-1, GSDMD, IL-1β and IL-18.

7. Use of cyanidin-3-O-glucoside in the preparation of a preparation for reducing the level of any liver cell pyroptosis pathway protein as claimed in claim 6.

8. A drug for treating / preventing liver damage caused by aflatoxin, characterized in that: Contains cyanidin-3-O-glucoside.

9. The drug according to claim 8, characterized in that It also includes pharmaceutically acceptable carriers to prepare different dosage forms.

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