Application of troxerutin in preparation of medicine for treating acute liver injury

Abstract

The invention provides an application of troxerutin in preparation of drugs for treating acute liver injury, the troxerutin can reduce ALT and AST levels in serum of a CCl4-induced acute liver injury mouse, and can inhibit expression of CCl4-induced acute liver injury inflammatory factors F4 / 80, CD68, CD11b and CD45, so that troxerutin is a potential drug for treating acute liver injury symptoms.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and relates to the application of troxerutin in preparing a medicine for treating acute liver injury. Background Art

[0002] Liver diseases can be categorized into two main categories: inflammatory liver disease and fatty liver disease. Inflammatory liver disease includes hepatitis and cirrhosis, with hepatitis further divided into acute and chronic hepatitis. Hepatitis can be caused by viruses, obesity, alcohol, and other factors. If left untreated, it can easily develop into chronic hepatitis and even cirrhosis.

[0003] Flavonoids are a class of natural products found widely in plants and have attracted significant attention for their diverse biological activities. In the treatment of liver diseases, flavonoids primarily alleviate liver inflammation through their antioxidant and anti-inflammatory properties, hepatocyte protection, lipid metabolism, and effects on liver fibrosis. Troxerutin is a flavonoid found in a variety of natural plants, such as ryegrass and grape seeds. Extracts from these plants are often rich in troxerutin and other bioactive components. To date, there have been no reports demonstrating that troxerutin can alleviate liver inflammation. Summary of the Invention

[0004] Based on the above content, the purpose of the present invention is to provide a use of troxerutin in the preparation of a drug for treating acute liver injury.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The mechanism of action of troxerutin in the preparation of drugs for the treatment of acute liver injury is to reduce the levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in the serum of mice with CCl4-induced acute liver injury, while inhibiting the expression of inflammatory factors F4 / 80, CD68, CD11b and CD45 in mice with CCl4-induced acute liver injury, thereby reducing the infiltration of acute liver injury into liver inflammation.

[0006] In this application, the dosage concentration of troxerutin is 20 mg / kg.

[0007] The present invention has the following beneficial effects: (1) Troxerutin can reduce the levels of ALT and AST in the serum of mice with acute liver injury induced by CCl4; (2) Troxerutin can inhibit the expression of inflammatory factors F4 / 80, CD68, CD11b and CD45 in acute liver injury induced by CCl4. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is the result of the effect of troxerutin on AST and ALT in the liver function of mice with acute liver injury; Figure 2 This figure shows the effect of troxerutin on the pathological structure of liver tissue in mice with acute liver injury; Figure 3 This figure shows the effect of troxerutin on the expression of inflammatory factors F4 / 80, CD68, CD11b and CD45 proteins in the liver tissue of mice with acute liver injury; Figure 4 This figure shows the effect of troxerutin on the mRNA expression of inflammatory factors F4 / 80, CD68, CD11b and CD45 in the liver tissue of mice with acute liver injury. DETAILED DESCRIPTION

[0009] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0010] The present invention provides an application of troxerutin, namely its application in the preparation of a drug for treating acute liver injury. Specific experiments are provided below to illustrate this application.

[0011] 1. Experimental Materials C57 male SPF mice, 8 weeks old, weighing 18–22 g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., license number SCXK (Lu) 20220006. They were fed a standard diet and housed in a 12-h light / 12-h dark cycle. Experiments were performed after six mice per cage had been allowed to acclimatize for one week. CCl4 (analytical grade, Shanghai Titan Technology Co., Ltd.), automatic biochemical analyzer (BM6010 / C), qPCR instrument (QuantStudio 6 Pro), PCR instrument (Eppendorf), reverse transcription kit (Vazyme, R223), CD45 antibody (Thermo Fisher Scientific, 48-0451-82), CD68 antibody (Bio-rad, MCA1957), CD11b antibody (eBioscience, 14-0112-82), F4 / 80 antibody (Abclonal, A23788), Donkey Anti-Rat 488 antibody (Jackson, 712-545-150), Donkey Anti-Rabbit 488 antibody (Jackson, 711-545-152).

[0012] 2. Experimental methods After 7 days of adaptive feeding, 18 C57 mice were randomly divided into three groups: a blank group, a model group, and a treatment group, with 6 mice in each group. The blank group received normal saline by gavage for 3 days, followed by two gavages of normal saline on the fourth day, with a 12-hour interval. Samples were collected on the fifth day. This group is referred to as the blank group (CON). The model group received normal saline by gavage for the first three days. On the fourth day, mice were intraperitoneally injected with 25% CCl₄ at a volume of 4 μL / g to establish an acute liver injury model. On the same day, normal saline was again gavaged twice, with a 12-hour interval. Samples were collected on the fifth day. This group is referred to as the model group (CCl₄). The treatment group received troxerutin by gavage for 3 days. On the fourth day, mice were intraperitoneally injected with 25% CCl₄ at a volume of 4 μL / g to establish an acute liver injury model. On the same day, troxerutin was again gavaged twice, with a 12-hour interval, at a concentration of 20 mg / kg. Samples were collected on the fifth day. This group is referred to as the treatment group (CCl₄+Troxerutin).

[0013] The blank group, model group and drug-treated group mice were subjected to serum liver function test, HE staining test, immunofluorescence staining test and inflammatory factor expression test. The specific test contents and test results are as follows: 1. Serum liver function test 24 hours after CCl4 injection, blood was collected from the eye sockets of mice in each group and placed in anticoagulant tubes. After the blood was placed at 4°C for 1 hour, it was centrifuged in a refrigerated centrifuge at 15000 rpm for 15 minutes at 4°C to separate the serum. The serum was collected and the levels of ALT and AST were measured using an automatic analytical biochemical analyzer. Figure 1 .

[0014] By the attached Figure 1 As can be seen, compared with the blank group, the serum AST and ALT levels of the model group mice were significantly increased, with a significant difference. Compared with the model group, the serum AST and ALT levels of the treated group mice were significantly decreased, with a significant difference. The results show that troxerutin can reduce the serum AST and ALT levels of mice with acute liver injury caused by CCl4, helping to alleviate liver damage in mice.

[0015] 2. HE staining The liver tissues of the mice in each group were taken and immersed in 4% PFA (paraformaldehyde). The subsequent paraffin embedding, sectioning and HE staining were carried out according to the existing methods. Figure 2 .

[0016] By the attached Figure 2As can be seen, the hepatocytes of the blank group mice were neatly arranged, the hepatic lobule structure was intact, and there was no inflammatory infiltration. The hepatocytes of the model group mice were disordered, and large areas of inflammatory infiltration were present. The area of inflammatory infiltration in the treated group mice was significantly reduced compared to the model group, indicating that troxerutin has a significant effect on improving the hepatic inflammatory infiltration of mice with acute liver injury caused by CCl4.

[0017] 3. Immunofluorescence staining F4 / 80, CD68, CD11b, and CD45 are four common inflammatory cell markers in the immune system. F4 / 80 is a marker for mouse macrophages, primarily expressed in mouse macrophages, and plays a role in macrophage recognition, adhesion, and function. CD68 is a macrophage-specific marker and a lysosome-associated protein. It is primarily used to identify macrophages and some dendritic cells and is important for the phagocytosis and clearance of intracellular and extracellular particles, dead cells, and pathogens. CD11b is part of the integrin family, expressed on immune cells and involved in cell adhesion, migration, and phagocytosis. CD45 is a leukocyte antigen, a cell surface protein that helps white blood cells recognize foreign antigens and participate in immune responses. It participates in signal transduction between T and B cells and also plays a crucial role in the activation and proliferation of immune cells. Under inflammatory conditions, immune cells release more cytokines, leading to increased expression of F4 / 80, CD68, CD11b, and CD45. Based on this, the examples of the present application respectively detected the protein expression levels of inflammatory factors F4 / 80, CD68, CD11b and CD45 in each group of mice.

[0018] Liver tissues from model and treatment groups were obtained and fixed in 4% PFA for 1 hour. Following fixation, the tissues were washed three times with 1X PBS (phosphate buffered saline), dehydrated in 30% sucrose solution for 10 hours, and finally embedded in OCT (optimal cutting temperature compound). After embedding, the liver tissues from both model and treatment groups were cut into 10-μm sections. After the sections were air-dried, a waterproof marker was used to mark the edges of the tissue to prevent subsequent staining. The sections were then soaked in PBS for 15 minutes to remove the OCT. Primary antibodies diluted in PBST (1X PBS + 0.2% Triton X-100) were applied to the sections and incubated overnight at 4°C in the dark. On the second day, the liver tissue sections of the mice in the model group and the drug-treated group were washed and soaked in 1X PBS for 15 minutes, and then incubated with secondary antibodies at room temperature for 30 minutes. After soaking again, the secondary antibodies were washed off and the sections were sealed with mounting media. Nail polish was applied on both ends to prevent slipping. Immunofluorescence staining was performed on the mice in the model group and the drug-treated group to obtain attached Figure 3 .

[0019] By the attached Figure 3 It can be seen that compared with the mice in the model group, the expression levels of F4 / 80, CD68, CD11b and CD45 in the treatment group were significantly reduced, which showed that troxerutin significantly reduced the expression levels of F4 / 80, CD68, CD11b and CD45 in the liver of mice with acute liver injury caused by CCl4, suggesting the anti-inflammatory effect of troxerutin in the acute CCl4 liver injury model.

[0020] 4. Detection of inflammatory factor expression Liver tissue from each modeled mouse was collected and added to 1 mL of Trizol. The tissue was thoroughly ground in liquid nitrogen. 200 μL of chloroform was then added, mixed by inversion, and allowed to stand on ice for 10 minutes. After this stand, the tube was centrifuged at 15,000 rpm at 4°C for 15 minutes. The upper aqueous phase was aspirated and transferred to another nuclease-free EP tube. An equal volume of pre-chilled isopropanol was added, mixed by inversion, and allowed to stand on ice for 10 minutes. After this stand, the tube was centrifuged at 15,000 rpm at 4°C for 15 minutes. The supernatant was discarded, and 1 mL of pre-chilled 75% ethanol (750 μL of anhydrous ethanol and 250 μL of DEPC water) was added to each tube. The mixture was mixed by inversion, and the tube was centrifuged at 7,500 rpm at 4°C for 5 minutes. The supernatant was discarded as much as possible, and after air drying, 20 μL of DEPC water was added to each tube. RNA concentration was measured after 5 minutes. The extracted RNA was reverse transcribed using a reverse transcription kit, and the added volume was calculated based on the RNA concentration.

[0021] Prepare the system by adding 2 μL of primers, 6 μL of DEPC, 10 μL of Mix, and 2 μL of extracted RNA to each well. Use an enzyme-free pipette tip to add the sample to a 96-well plate. Repeat for each sample in triplicate. After adding the sample, seal the 96-well plate with film and centrifuge at 4000 rpm for three minutes. After setting the conditions on the qPCR instrument, amplify the attached RNA. Figure 4 The mRNA expression levels of F4 / 80, CD68, CD11b, and CD45 are shown.

[0022] By the attached Figure 4As can be seen, compared with the blank group, the mRNA expression levels of F4 / 80, CD68, CD11b, and CD45 in the model group mice were significantly increased, with significant differences. Compared with the model group, the mRNA expression levels of F4 / 80, CD68, CD11b, and CD45 in the treatment group mice were significantly decreased, with significant differences. This shows that troxerutin can significantly reduce the expression levels of F4 / 80, CD68, CD11b, and CD45 in the acute CCl4 liver injury model, suggesting that troxerutin has an anti-inflammatory effect in the acute CCl4 liver injury model.

[0023] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of troxerutin in the preparation of drugs for the treatment of acute liver injury.

2. The use according to claim 1, characterized in that The troxerutin is used for reducing the ALT and AST levels in the serum of mice with acute liver injury induced by CCl4.

3. The use according to claim 1, characterized in that The troxerutin is used for inhibiting the expression of inflammatory factors F4 / 80, CD68, CD11b and CD45 in mice with acute liver injury induced by CCl4.

4. The use according to claim 1, characterized in that The dosage concentration of troxerutin is 20 mg / kg.

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