Use of fish swim bladder mucopolysaccharide in preparation of drug for liver injury

Abstract

Use of a fish swim bladder mucopolysaccharide in the preparation of a drug or dietary supplement for ameliorating liver injury. The fish swim bladder mucopolysaccharide can ameliorate pathological damages such as arsenic-induced hepatic sinusoid and central venous congestion, cytoplasmic vacuolization of liver cells, and inflammatory infiltration, can ameliorate arsenic-induced liver cell damage and necrosis, and liver tissue oxidative imbalance, and can also significantly up-regulate the relative expression level of antioxidation-related genes in liver tissues, down-regulate the relative expression level of detoxification-related genes, and alleviate arsenic-induced liver damage.

Description

Use of swim bladder mucopolysaccharide in preparation of liver injury drugs TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to use of swim bladder mucopolysaccharide in preparation of liver injury drugs or dietary supplements. BACKGROUND

[0002] Swim bladder, also known as fish glue, is a nourishing product with medicinal and edible properties, and is rich in various functional active substances. Main components include collagen, mucopolysaccharide, vitamins, and trace elements such as calcium, zinc, potassium, and selenium. Swim bladder mucopolysaccharide is a kind of extracellular biological macromolecule secreted by cells, and is the sugar chain part of the protein polysaccharide molecule in the animal body. It has the functions of regulating inorganic substances and water in extracellular fluid, promoting bone formation, promoting wound healing, preventing infection, lubricating cartilage, preventing thrombosis, purifying blood, and maintaining corneal transparency. The existing patent CN117018020A discloses that swim bladder glycosaminoglycan can slow down the occurrence of intestinal oxidative stress and inflammation, and has good application prospect in preparation of intestinal injury drugs.

[0003] Arsenic is a carcinogenic environmental pollutant widely existing in the earth's crust, soil and air. Due to the high accumulation capacity of aquatic organisms for arsenic, in recent years, dietary exposure has become a common route for arsenic to enter the body. Arsenic exposure can cause damage to multiple organs such as liver, kidney, intestine, nervous system and reproductive system. As the most important metabolic organ of arsenic, the liver has attracted widespread attention in the related research of arsenic-induced liver damage. Studies have shown that arsenic exposure can cause liver fibrosis, non-alcoholic fatty liver and liver cancer, and the causes are related to arsenic-induced oxidative stress, ferroptosis and autophagy.

[0004] Since arsenic-induced liver damage is often an accumulation process, there is usually no obvious phenotype in the early exposure stage, and acute damage begins to appear when it accumulates to a certain extent. As an auxiliary means of diet, dietary supplements are used to supplement the body's needs of amino acids, trace elements, vitamins, minerals, etc., and can be taken with meals. It can prevent nutritional deficiencies and reduce the risk of certain diseases. Therefore, it is of great significance to provide a dietary supplement for liver protection in the early exposure stage of arsenic, and it is also very important to provide a drug that can alleviate arsenic-induced liver damage. SUMMARY

[0005] To solve the above technical problems, the present application provides use of swim bladder mucopolysaccharide in preparation of liver injury drugs or dietary supplements. Swim bladder mucopolysaccharide can improve pathological damage such as liver sinusoidal congestion, hepatocyte vacuolization and inflammatory infiltration, can improve liver cell damage and necrosis, slow down oxidative damage of liver tissue, and regulate expression of toxicity-related genes in the body. The present application promotes the utilization of swim bladder resources and provides a method for alleviating arsenic-induced liver damage.

[0006] To achieve the technical purposes of the present application, in one aspect, the present application provides the use of fish swim bladder mucopolysaccharide in the preparation of a drug for liver injury, the liver injury being arsenic-induced liver injury, and the effective component of the fish swim bladder mucopolysaccharide being used in an amount of 50-200 mg / kg.

[0007] Further, the fish swim bladder mucopolysaccharide provided by the present application can alleviate the liver pathological injury caused by arsenic induction, specifically including: liver sinusoid and central vein hyperemia, lymphocyte small focal infiltration, and hepatocyte cytoplasm vacuolization. The present application observes the HE staining results of mouse liver sections and finds that the arsenic exposure model group can clearly see that the liver sinusoid and central vein are both hyperemic, accompanied by lymphocyte small focal infiltration and cytoplasm vacuolization in multiple hepatocytes; after 38 days of continuous gavage of 50-200 mg / kg of fish swim bladder mucopolysaccharide, the liver sinusoid hyperemia, central vein hyperemia, and lymphocyte small focal infiltration are all significantly improved, and the hepatocyte morphological structure is complete, and there is almost no cytoplasm vacuolization phenomenon.

[0008] Further, the fish swim bladder mucopolysaccharide provided by the present application can alleviate the liver cell injury and necrosis caused by arsenic induction, specifically including: increased liver cell ALT activity and increased liver cell AST activity. The present application determines the mouse liver cell injury indicators and finds that arsenic exposure can cause the liver cell ALT activity level to increase by 128.49%, the liver cell AST activity level to increase by 63.58%, and cause serious liver cell necrosis; after 38 days of continuous gavage of 50-200 mg / kg of fish swim bladder mucopolysaccharide, the ALT and AST levels in the mouse serum return to the levels of the control group, indicating that the fish swim bladder mucopolysaccharide can effectively improve arsenic-induced liver cell injury and necrosis.

[0009] Further, the fish swim bladder mucopolysaccharide provided by the present application can alleviate the liver tissue oxidation imbalance caused by arsenic induction, specifically including: decreased T-SOD activity and GSH content in the liver tissue. The present application determines the mouse liver tissue oxidation indicators and finds that arsenic exposure can cause the T-SOD activity and GSH content representing the antioxidant activity in the liver tissue to decrease significantly, with the decrease levels being 18.29% and 64.40% respectively, and can cause liver tissue oxidation imbalance; after 38 days of continuous gavage of 50-200 mg / kg of fish swim bladder mucopolysaccharide, the T-SOD activity and GSH content in the mouse liver tissue are both significantly improved, indicating that the fish swim bladder mucopolysaccharide can effectively improve liver tissue oxidation injury.

[0010] Further, the fish swim bladder mucopolysaccharide provided by the present application can significantly up-regulate the relative expression amount of antioxidant-related genes and down-regulate the relative expression amount of detoxification-related genes, and can effectively improve liver tissue oxidation injury. The antioxidant-related genes include Nrf2 gene, SOD-1 gene, and GPX-4 gene, and the detoxification-related genes include MT-1 gene.

[0011] Further, the swim bladder mucopolysaccharide provided by the present application can increase the relative expression amount of Nrf2 protein in liver tissue nuclear protein, and the swim bladder mucopolysaccharide significantly promotes the nuclear entry of Nrf2, activates the expression of endogenous antioxidant system, and can effectively play a role in slowing down the arsenic toxicity of the body.

[0012] In another aspect, the present application claims the use of the swim bladder mucopolysaccharide in the preparation of a dietary supplement for liver damage, and the liver damage is arsenic-induced liver damage. The dietary supplement is composed of the swim bladder mucopolysaccharide and a diet-acceptable auxiliary material, the diet-acceptable auxiliary material refers to an auxiliary material that retains the biological efficacy of the swim bladder mucopolysaccharide and has no adverse effects in biology or other aspects, and the diet-acceptable auxiliary material includes colorants, enzyme preparations, thickening agents, leavening agents, sweeteners, etc. In addition, the raw material composition selected by the present application can further contain natural extracts with antioxidant effects such as radix puerariae, honeysuckle, dietary fiber, tea polyphenols, etc. According to the known technology in the art, the dietary supplement can be prepared into various product forms such as oral liquids, tablets, capsules, biscuits, candies and beverages, etc. according to needs.

[0013] Compared with the prior art, the technical solution provided by the present application at least has the following beneficial effects or advantages:

[0014] The present application observes the HE staining results of mouse liver sections and finds that the liver sinus and central vein of the arsenic exposure model group can be obviously seen to appear hyperemia, accompanied by lymphocyte small focal infiltration and cytoplasm vacuoles in multiple hepatocytes; after continuous gavage of 50-200 mg / kg of swim bladder mucopolysaccharide for 38 days, the liver sinus hyperemia, central vein hyperemia and lymphocyte small focal infiltration are obviously improved, the hepatocyte morphological structure is complete, and the cytoplasm vacuole phenomenon is almost eliminated.

[0015] The present application finds that arsenic exposure can cause the ALT activity level of hepatocytes to increase by 128.49%, the AST activity level of hepatocytes to increase by 63.58%, and cause serious hepatocyte necrosis by determining the liver cell damage indicators of mice; after continuous gavage of 50-200 mg / kg of swim bladder mucopolysaccharide for 38 days, the ALT and AST levels in the serum of mice return to the enzyme levels of the control group, indicating that the swim bladder mucopolysaccharide can effectively improve arsenic-induced hepatocyte damage and necrosis.

[0016] The present application finds that arsenic exposure can cause the T-SOD activity and GSH content in liver tissue, which represent antioxidant activity, to decrease significantly by 18.29% and 64.40% respectively, and can cause liver tissue oxidation imbalance by determining the oxidation indicators of mouse liver tissue; after continuous gavage of 50-200 mg / kg of swim bladder mucopolysaccharide for 38 days, the T-SOD activity and GSH content in the liver tissue of mice are significantly improved, indicating that the swim bladder mucopolysaccharide can effectively improve the oxidative damage of liver tissue.

[0017] The present application finds that the swim bladder mucopolysaccharide can significantly up-regulate the relative expression amount of the antioxidant-related genes and down-regulate the relative expression amount of the detoxification ability genes by determining the relative expression amount of the antioxidant genes and the detoxification genes in the liver tissues of mice, and can effectively improve the oxidative damage of the liver tissues. The swim bladder mucopolysaccharide of the present application significantly promotes the nuclear entry of Nrf2, activates the expression of the endogenous antioxidant system, and can effectively play the role of slowing down the arsenic toxicity of the body. BRIEF DESCRIPTION OF DRAWINGS

[0018] Fig. 1 is the HE staining result of the liver sections of mice in different experimental groups.

[0019] Fig. 2 is the determination result of the liver cell damage of mice in different experimental groups. Fig. 2A is the determination result of the ALT activity of the liver cells of mice in different experimental groups; Fig. 2B is the determination result of the AST activity of the liver cells of mice in different experimental groups.

[0020] Fig. 3 is the determination result of the oxidative indexes of the liver tissues of mice in different experimental groups. Fig. 3A is the determination result of the T-SOD activity of the liver tissues of mice in different experimental groups; Fig. 3B is the determination result of the GSH content of the liver tissues of mice in different experimental groups.

[0021] Fig. 4 is the determination result of the relative expression amount of the toxicity alleviating genes in the liver tissues of mice in different experimental groups. Fig. 4A is the determination result of the relative expression amount of the Nrf2 gene in the liver tissues of mice in different experimental groups; Fig. 4B is the determination result of the relative expression amount of the SOD-1 gene in the liver tissues of mice in different experimental groups; Fig. 4C is the determination result of the relative expression amount of the GPX-4 gene in the liver tissues of mice in different experimental groups; Fig. 4D is the determination result of the relative expression amount of the MT-1 gene in the liver tissues of mice in different experimental groups.

[0022] Fig. 5 is the determination result of the relative expression amount of the Nrf2 protein in the nuclear proteins of the liver tissues of mice in different experimental groups. Fig. 5A is the electrophoresis strip chart of the relative expression amount of the Nrf2 protein in the nuclear proteins of the liver tissues of mice in different experimental groups; Fig. 5B is the statistical result chart of the relative expression amount of the Nrf2 protein in the nuclear proteins of the liver tissues of mice in different experimental groups. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be described in combination with the embodiments, but the present application is not limited to the following embodiments. The experimental methods and detection methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. EMBODIMENT

[0024] The present embodiment provides an animal test of the improvement effect of fish swim bladder mucopolysaccharide on arsenic-induced liver injury in mice. The preparation method of fish swim bladder mucopolysaccharide is the same as that in Example 1 of patent CN112107590B. The fish swim bladder mucopolysaccharide is a chondroitin sulfate A analogue containing a→4GlcUAβ1→3GalNAc(4S)β1→ main chain.

[0025] 1. Building a test model

[0026] 60 male SPF level C57BL / 6J mice (18±2g, 7 weeks old) were randomly divided into 5 groups (control group, model group, low-dose group, medium-dose group and high-dose group), 12 in each group. The test groups were divided into three groups according to the intragastric dose of fish swim bladder mucopolysaccharide, including low-dose group, medium-dose group and high-dose group. The intragastric dose of the low-dose group was 50mg / kg, the intragastric dose of the medium-dose group was 100mg / kg, and the intragastric dose of the high-dose group was 200mg / kg. The test groups were given fish swim bladder mucopolysaccharide solution in the morning and 5mg / kg sodium arsenite solution in the afternoon; the model group was given 0.1mL / 10g of normal saline in the morning and 5mg / kg of sodium arsenite solution in the afternoon; the control group was given 0.1mL / 10g of normal saline in the morning and in the afternoon. After the intragastric administration of each group of mice was completed every day, the mice were allowed to freely eat, and the experimental groups (control group, model group, low-dose group, medium-dose group and high-dose group) were continuously given continuous intragastric administration for 38 days. After the last intragastric administration, the mice were sacrificed, and the eyeball serum and liver tissue were collected.

[0027] 2. Pathological observation of mouse liver

[0028] The same part of the largest lobe of the mouse liver was fixed in 4% paraformaldehyde, and after paraffin embedding, sectioning and H&E staining, the tissue morphology was observed under an optical microscope (×200), as shown in Figure 1.

[0029] As can be seen from Figure 1, compared with the control group, the arsenic exposure model group can clearly see that the liver sinusoids and central veins are congested (circled), accompanied by lymphocyte focal infiltration (rectangular) and multiple liver cells with cytoplasmic vacuoles (arrow). After the action of fish swim bladder mucopolysaccharide, the mouse liver has improved to varying degrees, showing that the liver cell morphology is complete and almost no cytoplasmic vacuoles; the central vein congestion and inflammatory infiltration are significantly improved.

[0030] 3. Determination of basic indicators of mouse liver injury

[0031] The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum (liver cell damage determination) and the total superoxide dismutase (T-SOD) activity and glutathione (GSH) content in liver tissue (liver tissue oxidation determination) were determined according to the instructions of the detection kit (all purchased from Nanjing Jiancheng Biological Engineering Institute). The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum are shown in Figure 2, and the total superoxide dismutase (T-SOD) activity and glutathione (GSH) content in mouse liver tissue are shown in Figure 3.

[0032] As can be seen from Figure 2, compared with the control group, the activities of ALT and AST in the serum of the arsenic exposure model group, which represent liver cell damage and necrosis, were significantly increased, with an increase of 128.49% and 63.58%, respectively, indicating that arsenic exposure caused severe liver cell necrosis. After the action of fish swim-bladder mucopolysaccharide at various doses, the release of the two enzymes was significantly reduced, and the levels of ALT and AST in mouse serum returned to the enzyme levels of the control group, indicating that fish swim-bladder mucopolysaccharide can effectively improve arsenic-induced liver cell damage and necrosis. At the ALT activity level, the medium-dose group was the most optimal, and the ALT activity was significantly reduced by 46.97% compared with the model group. At the AST activity level, the high-dose group was the most optimal, and the AST activity was reduced by 55.53% compared with the model group.

[0033] As can be seen from Figure 3, compared with the control group, the T-SOD activity and GSH content in the liver tissue of the arsenic exposure model group, which represent antioxidant activity, were significantly reduced, with a decrease of 18.29% and 64.40%, respectively, indicating that arsenic exposure can cause oxidative imbalance in liver tissue. After the action of fish swim-bladder mucopolysaccharide, the T-SOD activity and GSH content were significantly improved, indicating that fish swim-bladder mucopolysaccharide can effectively improve liver tissue oxidative damage. Among them, the T-SOD activity of the liver tissue of the mouse in the high-dose group was slightly higher than that of the control group, indicating that fish swim-bladder mucopolysaccharide has good antioxidant activity; at the GSH content level, the medium-dose group was the most optimal, and the GSH content of the mouse liver tissue was significantly higher than that of the control group, and was 4.39 times that of the model group.

[0034] 4. Determination of the Relative Expression Amount of Mouse Liver Toxicity Alleviation Genes

[0035] RNA was extracted from mouse liver tissue using an RNA extraction kit, and cDNA was obtained by reverse transcription, followed by the addition of an RNA fluorescent quantitative reagent. The relative expression amounts of Nrf2 gene, SOD-1 gene, GPX-4 gene and MT-1 gene were determined by RT-qPCR, and the internal reference gene was GADPH. The calculation formula was 2 –ΔΔCtNrf2 gene maintains the intracellular redox balance by regulating the expression of a series of antioxidant enzymes, protects cells from oxidative stress damage, and is an antioxidant-related gene; Cu / Zn superoxide dismutase 1 (SOD-1) is an antioxidant-related gene; GPX-4 gene is an antioxidant-related gene in mouse liver tissue; metallothionein (MT) is an endogenous cell protector that can resist liver toxicity by scavenging free radicals and antioxidant, and MT-1 gene is a detoxification capacity-related gene in mouse liver tissue. The relative expression amounts of Nrf2 gene, SOD-1 gene, GPX-4 gene and MT-1 gene in mouse liver tissue are shown in FIG. 4.

[0036] As can be seen from FIG. 4, compared with the control group, the Nrf2 gene, SOD-1 gene and GPX-4 gene representing antioxidant capacity in the liver tissue of the arsenic exposure model group all showed a downward trend, among which the Nrf2 gene and GPX-4 gene showed a significant downward level; the relative expression amount of MT-1 gene representing detoxification capacity increased significantly. The downward level of the relative expression amount of Nrf2 gene was 36.01%, the downward level of the relative expression amount of SOD-1 gene was 14.83%, the downward level of the relative expression amount of GPX-4 gene was 59.42%, and the relative expression amount of MT-1 gene increased by 9.60 times, indicating that arsenic exposure can cause serious oxidative damage to mouse liver. After the action of swim bladder mucopolysaccharide, the relative expression amounts of antioxidant Nrf2 gene, SOD-1 gene and GPX-4 gene were significantly up-regulated, and the relative expression amount of MT-1 gene was down-regulated. Among them, the high-dose group of swim bladder mucopolysaccharide had the best improvement effect, and the up-regulation effects of Nrf2 gene, SOD-1 gene and GPX-4 gene relative expression amount compared with the model group were 100.39%, 99.68% and 211.44% respectively, which could significantly up-regulate the genes representing antioxidant capacity; the high-dose group could also significantly down-regulate the relative expression amount of MT-1 gene, which was down-regulated by 46.92% compared with the model group. The results show that swim bladder mucopolysaccharide can effectively improve the oxidative damage of liver tissue.

[0037] 5. Determination of the relative expression amount of Nrf2 protein in the nuclear protein of mouse liver tissue

[0038] Nrf2 protein binds to the antioxidant response element in the promoter region of the cell protection gene, which is an oxidative stress-related transcription factor. According to the related steps of the nuclear protein extraction kit (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.), the nuclear protein in the liver tissue was extracted, and then the protein quantitative kit (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) was used to determine the concentration of each sample. After adjusting the same, 5x SDS-PAGE protein loading buffer was added, and then the protein was denatured at 100℃ for 10min. The protein was separated by SDS-PAGE electrophoresis, and then transferred to a PVDF membrane. After blocking at room temperature for 1h, the primary antibody LaminB1 and Nrf2 (antibody dilution ratio was 1:1000) were incubated at 4℃ overnight, and then the corresponding secondary antibody (dilution ratio was 1:3000) was incubated at room temperature for 1h, and then soaked in hypersensitive ELC working solution. The chemical luminescence system was used for imaging, and the obtained protein band image was analyzed by Image J software. The final data was presented as the ratio of the gray value of the target protein to the gray value of the internal reference LaminB1. The determination results of the relative expression amount of Nrf2 protein in the nuclear protein of mouse liver tissue are shown in Figure 5.

[0039] As shown in Figure 5, compared with the control group, the Nrf2 level in the liver tissue nucleus of the arsenic exposure model group increased, indicating that arsenic induced oxidative stress. After the action of fish swim bladder mucopolysaccharide, each dose group significantly promoted the nuclear import of Nrf2 and activated the expression of endogenous antioxidant system. Among them, the activation degree was the highest under the action of the medium dose. The results show that fish swim bladder mucopolysaccharide is an Nrf2 activator, which can effectively activate the antioxidant system of the body and play a role in slowing down the toxicity of arsenic.

[0040] As described above, the basic principles, main features and advantages of the present application are better described. The above examples and descriptions only describe the preferred embodiments of the present application, and the present application is not limited by the above examples. Without departing from the spirit and scope of the present application, various changes and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the scope of protection of the present application.

Claims

1. Use of fish swim-bladder mucopolysaccharides for the preparation of a medicament for the treatment of liver damage, characterized in that, The liver injury is arsenic-induced liver injury.

2. Use of the swim bladder mucopolysaccharide according to claim 1 in the preparation of a drug for liver damage, characterized in that, The effective component of the fish swim bladder mucopolysaccharide is used in an amount of 50-200 mg / kg.

3. Use of the swim bladder mucopolysaccharide according to claim 1 in the preparation of a drug for liver damage, characterized in that, The fish swim bladder mucopolysaccharide alleviates the arsenic-induced liver pathological injury. The arsenic-induced liver pathological injury includes liver sinus and central vein hyperemia, lymphocyte small focal infiltration and hepatocyte cytoplasm vacuolization.

4. The use of the swim bladder mucopolysaccharide according to claim 1 in the preparation of a drug for liver damage, characterized in that, The fish swim bladder mucopolysaccharide alleviates arsenic-induced hepatocyte injury and necrosis. The arsenic-induced hepatocyte injury and necrosis includes hepatocyte ALT activity increase and hepatocyte AST activity increase.

5. The use of the swim bladder mucopolysaccharide according to claim 1 in the preparation of a drug for liver damage, characterized in that, The fish swim bladder mucopolysaccharide alleviates arsenic-induced liver tissue oxidative imbalance. The arsenic-induced liver tissue oxidative imbalance includes liver tissue T-SOD activity decrease and GSH content decrease.

6. The use of the swim bladder mucopolysaccharide according to claim 1 in the preparation of a drug for liver damage, characterized in that, The fish swim bladder mucopolysaccharide up-regulates the relative expression amount of antioxidant-related genes in liver tissue and down-regulates the relative expression amount of detoxification-related genes. The antioxidant-related genes include Nrf2 gene, SOD-1 gene and GPX-4 gene. The detoxification-related genes include MT-1 gene.

7. Use of the swim bladder mucopolysaccharide according to claim 1 in the preparation of a drug for liver damage, characterized in that, The fish swim bladder mucopolysaccharide increases the relative expression amount of Nrf2 protein in liver tissue nuclear protein.

8. Use of fish swim bladder mucopolysaccharides for the preparation of a dietary supplement for liver damage, characterized in that, The liver injury is arsenic-induced liver injury.

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