Application of preparation of river clam polysaccharide in treatment of alcoholic liver injury by regulating gut-liver axis

Abstract

The application of mussel polysaccharide in preparation of a medicine for regulating the intestine-liver axis to treat alcoholic liver injury belongs to the technical field of natural products and biological medicine. The method takes mussel polysaccharide as an active ingredient, and divides experimental mice into a normal control group, an alcoholic liver injury model group, a positive control group and mussel polysaccharide low-, medium- and high-dose groups (60, 120 and 180 mg / kg / day), and continuously intervenes for 4 weeks. The results show that the mussel polysaccharide can significantly reduce liver function indexes such as ALT and AST, improve TC, TG, LDL-C, HDL-C related blood lipid disorders, protect the colon mucosal barrier, regulate the intestine-liver axis homeostasis, effectively reduce alcohol-induced liver fatty degeneration, inflammatory infiltration and oxidative stress injury, and the effect shows obvious dose dependence. The mussel polysaccharide is safe, non-toxic, economical in source, simple in intervention method and strong in repeatability, and provides a new way for the development of functional food and liver protection products for preventing and treating alcoholic liver injury.

Description

Technical Field

[0001] This invention belongs to the fields of food science and biomedicine, specifically relating to the application of freshwater mussel polysaccharide in improving alcoholic liver injury, and more particularly to the application of freshwater mussel polysaccharide in the preparation of drugs that regulate the gut-liver axis for the treatment of alcoholic liver injury. Background Technology

[0002] Alcoholic liver injury is a prevalent liver disease worldwide. Long-term excessive alcohol consumption can lead to fatty liver, alcoholic hepatitis, and liver fibrosis, and in severe cases, it can progress to cirrhosis and even liver cancer, posing a serious threat to human health. Currently, commonly used hepatoprotective drugs have problems such as significant side effects, limited availability, and high prices, making it difficult to meet the needs of long-term prevention and adjuvant therapy.

[0003] Marine and shellfish polysaccharides have become a research hotspot in the fields of biomedicine and functional foods due to their wide availability, high safety, and diverse biological activities. Freshwater mussels, widely farmed in my country, generate a large amount of meat byproducts (such as viscera and scraps) during their farming and processing. These byproducts are often discarded directly, causing not only resource waste but also environmental pollution.

[0004] In the existing technology, there is limited research on the extraction and application of freshwater mussel polysaccharides, and few reports on their use in improving alcoholic liver injury and regulating the gut-liver axis, indicating a significant technological gap. Therefore, this invention aims to discover a safe, efficient, and widely available natural polysaccharide from freshwater mussel by-products, providing a specific method for improving alcohol-induced liver and intestinal damage, filling this technological gap, and simultaneously realizing the resource utilization of freshwater mussel by-products. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of freshwater mussel polysaccharide in the preparation of a drug for regulating the gut-liver axis and treating alcoholic liver injury.

[0006] The mussel polysaccharide is derived from a byproduct of pearl mussel meat and is obtained through separation and purification.

[0007] The experimental procedure includes the following steps:

[0008] (1) Animal grouping and administration:

[0009] In this study, healthy experimental mice were selected and randomly divided into a normal control group, an alcoholic liver injury model group, a positive drug control group, and low, medium, and high concentration groups of freshwater mussel polysaccharide. The model group and each intervention group first established an alcoholic liver injury model by gavage with ethanol. After successful modeling, each intervention group was administered the drug by gavage at the corresponding concentration, while the normal control group and the model group were given an equal volume of physiological saline. The intervention was carried out for a preset period of time. The relevant norms of laboratory animal welfare ethics were followed during the experiment.

[0010] (2) Serum collection:

[0011] After the experiment, blood was collected from each group of mice, serum was separated by centrifugation, and stored at low temperature for subsequent detection of serum biochemical indicators.

[0012] (3) Preparation of liver and colon tissue homogenates:

[0013] Mouse liver and colon tissues were taken separately, added to physiological saline, homogenized in an ice bath, centrifuged, and the supernatant was collected and stored at low temperature for subsequent tissue biochemical index detection.

[0014] (4) Histopathological analysis of liver and colon tissues:

[0015] After fixing, dehydrating, embedding, and sectioning the liver and colon tissues, they were stained accordingly. The histopathological morphology was observed under a microscope to analyze liver damage and the integrity of the colonic mucosal barrier.

[0016] (5) Measurement of biochemical parameters:

[0017] The levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum and liver tissue were measured using a kit to clarify the intervention effect of freshwater mussel polysaccharide.

[0018] The present invention also provides a drug for regulating the gut-liver axis to treat alcoholic liver injury, wherein the main component of the drug that exerts the effect of treating alcoholic liver injury includes freshwater mussel polysaccharide.

[0019] The beneficial effects of this invention are:

[0020] (1) The raw materials are safe, cheap and widely available: the polysaccharide of freshwater mussels comes from the by-products of freshwater mussel processing, realizing the resource utilization of waste, reducing production costs, and the natural polysaccharide is safe, non-toxic and has no side effects, making it suitable for long-term use.

[0021] (2) Significant improvement effect and clear target: Mussel polysaccharide can significantly reduce the levels of ALT and AST in serum and liver tissue, improve the dyslipidemia state such as alcohol-induced TC, TG, LDL-C elevation and HDL-C reduction; at the same time, it can reduce liver steatosis, inflammatory infiltration and oxidative stress, protect the integrity of colonic mucosal barrier, and exert multi-target and multi-pathway liver protection by regulating the gut-liver axis. The improvement effect shows obvious concentration dependence. Attached Figure Description

[0022] Figure 1 The changes in biochemical indicators in the serum of mice in each group after modeling as described in Example 2. Figure 1A represents the change in total cholesterol (TC) levels in the serum of mice in each group after modeling. Figure 1 B represents the changes in serum triglyceride (TG) levels in mice of different groups after modeling. Figure 1 C represents the change in low-density lipoprotein cholesterol (LDL-C) levels in the serum of mice in each group after modeling. Figure 1 D represents the change in high-density lipoprotein cholesterol (HDL-C) levels in the serum of mice in each group after modeling.

[0023] Figure 2 The images show the pathological observation results of H&E staining of liver tissue, AB staining of colon tissue, and PAS staining of colon tissue in mice of each group after modeling as described in Examples 3 and 5.

[0024] Figure 3 The changes in biochemical indicators in the livers of mice in each group after modeling as described in Example 3. Figure 1 A represents the changes in aspartate aminotransferase (AST) levels in the liver tissues of mice in each group after modeling. Figure 1 B represents the changes in alanine aminotransferase (ALT) levels in the liver tissues of mice in each group after modeling. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0026] Example 1: Laboratory Animals and Intervention Program

[0027] The experimental animals and intervention implementation plan were carried out in accordance with the "(1) Animal grouping and administration" section of the invention. Specifically, as follows:

[0028] Experimental materials: SPF-grade male BALB / c mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., production license number: SCXK (Liaoning) 2020-0001, quality certificate number: 210726251100430172; simvastatin (positive control drug), freshwater mussel polysaccharide; relevant detection kits; all reagents used in the experiment were of analytical grade.

[0029] Animal feeding and grouping: Mice were housed in an SPF-grade cleanroom with a temperature controlled at 21-25℃ and a relative humidity maintained at 50-55%. They were provided with sterilized feed and distilled water. After one week of acclimatization, they were randomly divided into 6 groups of 6 mice each: normal control group, alcoholic liver injury model group, positive drug group (simvastatin 50 mg / kg·day), low concentration group of freshwater mussel polysaccharide (60 mg / kg·day), medium concentration group of freshwater mussel polysaccharide (120 mg / kg·day), and high concentration group of freshwater mussel polysaccharide (180 mg / kg·day).

[0030] Model construction and intervention: Except for the normal control group, all other groups of mice were administered 0.3 mL of 40% ethanol by gavage for 2 consecutive weeks to establish an alcoholic liver injury model. After successful model establishment, each group was administered the above concentration by gavage for 2 consecutive weeks. The normal control group and the model group were given the same volume of physiological saline by gavage once a day.

[0031] Sample collection: After the intervention, the mice were fasted for 12 hours, weighed, and then blood was collected from the eyeballs. The mice were dissected and the liver and colon tissues were separated for serum biochemical detection, tissue homogenate preparation and pathological analysis, respectively.

[0032] Table 1. Changes in body weight of mice in each group during the modeling process.

[0033] Group Days normal group Model group Positive drug group Low concentration polysaccharide group medium concentration polysaccharide group High concentration polysaccharide group 0 24.36±0.3 23.59±0.71 22.53±0.42 23.49±0.75 23.41±0.54 24.55±0.71 5 25.3±0.17 22.73±2.22 21.36±1.59 22.46±2.2 23.01±0.48 23.1±1.3 10 26.19±0.09 21.75±0.48 22.34±0.59 23.43±1.8 23.95±0.3 23.47±1.18 15 26.98±0.15 22.22±0.81 23.09±0.24 23.23±1.76 23.83±0.28 24.3±1.28 20 28.3±0.42 23.11±0.9 24.47±0.58 24.85±1.38 24.67±0.4 24.52±1.1 25 28.43±0.29 23.32±0.75 25.05±1.02 24.94±1.55 25.78±0.87 26.18±0.61 30 29.63±0.34 25.42±0.59 26.48±1.38 26.4±0.25 27.21±1.26 27.55±0.21

[0034] Note: Mouse body weight is measured in grams (g).

[0035] The results are shown in Table 1. Compared with the normal group, the body weight of mice in the model group was significantly reduced after 2 weeks of alcohol gavage, with a reduction range of 8.71%-11.57%, indicating that alcohol intake had caused damage to the mice. After polysaccharide intervention starting from week 3, the body weight of mice in all polysaccharide concentration groups showed a significant recovery. At the end of the intervention experiment, the average body weight of mice in the model group was the lowest, while the average body weight of mice in the high-polysaccharide group was the highest, approaching the level of the model group. These results indicate that polysaccharides can effectively slow down the decrease in body weight in mice with alcoholic liver injury and improve their overall health.

[0036] Example 2: Effects of freshwater mussel polysaccharides on serum biochemical indicators

[0037] The implementation plan for the effect of freshwater mussel polysaccharides on serum biochemical indicators shall be carried out in accordance with the "(2) Serum Collection" and "(5) Measurement of Biochemical Parameters" sections of the invention. Specifically, as follows:

[0038] Serum sample processing and detection: The collected blood was placed in a 1.5 mL centrifuge tube and centrifuged at 4℃ and 3000 r / min for 10 min. The supernatant was collected as serum, aliquoted and stored at -80℃. The levels of TC, TG, LDL-C and HDL-C in the serum were measured using the corresponding kit and in accordance with the kit instructions.

[0039] The results showed that, compared with the model group, the serum total cholesterol (TC) level in the model group mice was significantly increased by 100.8%. Compared with the model group, the serum TC levels in the normal group, positive drug group, and each polysaccharide concentration group were significantly decreased, with reductions of 47.83%, 12.02%, 18.32%, and 20.42%, respectively. The serum TG and LDL-C levels in the model group mice were significantly higher than those in each polysaccharide concentration group. The TG and LDL-C levels in each polysaccharide concentration group decreased in a concentration-dependent manner, gradually approaching the levels in the positive drug group and the model group. Compared with the model group, the serum HDL-C levels in each polysaccharide group were significantly increased, with increases of 5.93%, 14.07%, and 79.48%, respectively, with the high-polysaccharide group showing the best improvement. These results indicate that freshwater mussel polysaccharides have significant lipid-lowering activity and can effectively improve alcohol-induced dyslipidemia.

[0040] Example 3: Effects of freshwater mussel polysaccharides on liver pathology and biochemical indicators

[0041] The method for investigating the effects of freshwater mussel polysaccharides on liver pathology and biochemical indicators is as described in sections "(3) Preparation of liver and colon tissue homogenates", "(4) Pathological analysis of liver and colon tissues", and "(5) Measurement of biochemical parameters" in the invention. Specifically, the following steps are taken:

[0042] Liver tissue processing and testing: A portion of liver tissue was taken, homogenized according to the method described in the invention, and the supernatant was collected after centrifugation and stored at -80℃. The ALT and AST levels in the liver tissue were measured using a kit. Another portion of liver tissue was taken, fixed with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, and cut into 5 μm thick sections for H&E staining.

[0043] H&E staining: Liver samples were fixed in 4% paraformaldehyde for 24 hours, then dehydrated in ethanol, embedded in paraffin, and cut into 5 μm thick sections. The sections were stained with hematoxylin and eosin (H&E) sequentially. Finally, histopathological images were obtained using an inverted microscope to observe organ damage in the mouse liver as indicated by H&E.

[0044] In liver pathological observation, H&E staining results showed that the liver of the model group mice had obvious lipid accumulation, accompanied by severe steatosis and inflammatory cell infiltration. After intervention with freshwater mussel polysaccharide or simvastatin, the liver damage of mice was significantly restored, hepatocyte vacuoles were reduced, steatosis was alleviated, and inflammatory infiltration was improved. Moreover, the recovery effect of the high polysaccharide group was better than that of the low concentration polysaccharide group and the medium concentration polysaccharide group, and approached the level of the positive drug group.

[0045] Liver biochemical indicators: Compared with the normal group, the AST and ALT levels in the liver of mice in the model group were significantly increased by 145% and 36.78%, respectively, indicating that alcohol intake caused severe damage to hepatocytes; each concentration of freshwater mussel polysaccharide could significantly reduce the AST level in liver tissue, while the ALT level in liver tissue only showed a slight decrease, further proving that freshwater mussel polysaccharide has a significant effect on alcoholic liver injury.

[0046] Example 4: Effects of freshwater mussel polysaccharides on the colonic mucosal barrier and gut-hepatic axis homeostasis

[0047] The implementation plan for studying the effects of freshwater mussel polysaccharides on the colonic mucosal barrier and gut-hepatic axis homeostasis is as follows: (3) Preparation of liver and colon tissue homogenates and (4) Pathological analysis of liver and colon tissues in the invention. Specifically:

[0048] Colon tissue processing and detection: Mouse colon tissue was collected, and after measuring the colon length, some colon tissue was fixed with 4% paraformaldehyde at room temperature for 24 h, embedded in paraffin and sectioned (4 μm thick), and stained with alicin blue (AB) and periodic acid-chev (PAS) respectively.

[0049] (1) Alcian blue (AB) staining of colon tissue

[0050] Sectioning: Colon tissue blocks were sectioned using a paraffin microtome to a thickness of 4 mm.

[0051] Dewaxing to water: Soak in xylene for 40 min, anhydrous ethanol for 5 min, 85% ethanol for 5 min and 75% ethanol for 5 min in sequence, and then wash with purified water.

[0052] Alixin Blue Staining: Stain colon tissue sections with Alixin Blue solution for 15 min, then wash with purified water.

[0053] Dehydration, clearing and mounting: Dehydrated in sequence with 85% ethanol for 5 min, 95% ethanol for 5 min, and anhydrous ethanol for 10 min, then soaked in xylene for 10 min; finally mounted with neutral resin.

[0054] Microscopic observation: Observe and photograph under an optical microscope.

[0055] (2) Periodic acid-Chevrodysporine (PAS) staining of colon tissue

[0056] Sectioning: Colon tissue blocks were sectioned using a paraffin microtome to a thickness of 4 mm.

[0057] Dewaxing to water: Soak in xylene for 40 min, anhydrous ethanol for 5 min, 85% ethanol for 5 min and 75% ethanol for 5 min in sequence, and then wash with purified water.

[0058] Periodic acid staining: Stain colon tissue sections with periodic acid solution for 15 min, then wash with purified water.

[0059] Chev staining: Stain colon tissue sections with Chev stain in the dark for 30 min, then wash with purified water.

[0060] Hematoxylin staining: stain with hematoxylin solution for 5 min, wash with purified water; differentiate with differentiation solution for 30 s, wash with purified water; blue with blueing solution, wash with purified water.

[0061] Dehydration, clearing and mounting: Dehydrate with 85% ethanol for 5 min, 95% ethanol for 5 min, and anhydrous ethanol for 10 min in sequence; then soak in xylene for 10 min; finally mount with neutral resin.

[0062] Microscopic observation: The structure of the colonic mucosa and mucus secretion were observed and photographed under an optical microscope.

[0063] The remaining colon tissue was homogenized according to the method described in the invention and stored at low temperature for later use.

[0064] AB and PAS staining results showed that the content of acidic mucopolysaccharides and glycogen in the colonic mucosa of model group mice was significantly reduced, while the content of acidic mucopolysaccharides and glycogen in the colonic mucosa was significantly increased in each polysaccharide concentration group, with the high polysaccharide group showing the best effect. This indicates that freshwater mussel polysaccharides can maintain intestinal homeostasis by protecting the colonic mucosal barrier function. Therefore, based on the histopathological results of liver and colon tissues, it can be concluded that the high polysaccharide concentration group can more effectively improve the histopathological damage of liver and colon tissues in mice with alcoholic liver injury, reduce inflammatory cell infiltration, and protect gut-hepatic axis homeostasis.

[0065] In summary, the experimental results indicate that freshwater mussel polysaccharides can effectively improve alcohol-induced abnormal body weight in mice, significantly reduce ALT and AST levels in liver tissue, and improve lipid disorders such as TC, TG, LDL-C, and HDL-C in serum. Histopathological results further confirm that the polysaccharides can significantly improve hepatic steatosis and inflammatory infiltration in mice, while protecting the structural integrity of the colonic mucosa and maintaining intestinal barrier function. They exert multi-target, multi-pathway hepatoprotective effects by regulating the gut-liver axis, and the overall effect shows a certain concentration dependence. Therefore, polysaccharides have significant effects in improving alcoholic liver injury, regulating lipid metabolism, and protecting the intestinal mucosa, and possess great potential for development into drugs, functional foods, or hepatoprotective products for the prevention and treatment of alcoholic liver injury.

Claims

1. Preparation of freshwater mussel polysaccharides for the application of drugs that regulate the gut-liver axis in the treatment of alcoholic liver injury.

2. The application according to claim 1, characterized in that: The dosage of the freshwater mussel polysaccharide was 60 mg / kg / day to 180 mg / kg / day.

3. The application according to claim 1, characterized in that: The mussel polysaccharide is derived from a byproduct of pearl mussel meat and is obtained through separation and purification.

4. A drug for regulating the gut-liver axis to treat alcoholic liver injury, characterized in that: The main component in the drug that exerts its therapeutic effect on alcoholic liver injury includes freshwater mussel polysaccharide.

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