Application of American ginseng glycoprotein extract in the preparation of drugs for treating alcoholic liver disease

Abstract

This invention discloses the application of American ginseng glycoprotein extract in the preparation of drugs for treating alcoholic liver disease, belonging to the field of biomedical technology. This invention verifies that TD can significantly reduce serum ALT, AST, and TG levels in ALD mice, effectively reducing liver damage and lipid accumulation; inhibit NLRP3 protein expression and its downstream targets, effectively alleviating liver inflammation and pyroptosis; and improve intestinal flora imbalance in ALD mice. American ginseng glycoprotein extract has multi-target protective effects against alcoholic liver disease, reducing alcohol-induced liver tissue inflammation and oxidative stress damage. It has a mild composition and high safety profile, and can synergistically exert effects in liver protection, anti-inflammation, and antioxidation, making it suitable for adjunctive intervention and long-term management of alcoholic liver disease, providing a natural, mild, and effective protective pathway for alcoholic liver damage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of American ginseng glycoprotein extract in the preparation of drugs for treating alcoholic liver disease. Background Technology

[0002] Alcoholic liver disease (ALD) is the most common liver-related disease, characterized by high morbidity and mortality. It manifests as hepatic steatosis, oxidative stress, acetaldehyde toxicity, inflammation induced by various chemokines and cytokines, weakened intestinal barrier function, and dysbiosis. NOD-like receptor protein 3 (NLRP3) plays a crucial role in inflammatory diseases, including NLRP3, ASC, and Caspase-1. Upon stimulation by injury signals, inflammasomes activate Caspase-1, promoting the maturation of IL-18 and IL-1β. Simultaneously, activated Caspase-1 cleaves GSDMD, exposing its N-terminus and causing cell membrane perforation, thus inducing pyroptosis. Studies have shown that NLRP3 knockout mice exhibit protective effects against liver inflammation and steatosis under acute / chronic alcohol-feeding conditions.

[0003] American ginseng glycoprotein extract (TD) is an active ingredient derived from American ginseng, possessing both polysaccharide and protein structures. Its main functions include: antioxidation (scavenging free radicals, reducing oxidative damage, and delaying cell aging); immune regulation (activating immune cells, promoting antibody production, and enhancing the body's resistance); liver protection (reducing liver damage and assisting in maintaining liver function); anti-fatigue (relieving physical and mental fatigue and improving stress); and it can also help regulate blood sugar and lipids, protecting the cardiovascular system. It is widely used in the development of health foods and pharmaceuticals. Currently, no studies have been reported on the effects of TD on the NLRP3 signaling pathway and gut microbiota in ALD mice. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide the application of American ginseng glycoprotein extract in the preparation of a drug for treating alcoholic liver disease.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides the use of American ginseng glycoprotein extract in the preparation of drugs for treating alcoholic liver disease.

[0007] Based on the above technical solution, the American ginseng glycoprotein extract is further obtained by extracting American ginseng as raw material.

[0008] Based on the above technical solution, the drug further includes an effective amount of American ginseng glycoprotein extract and pharmaceutically acceptable excipients.

[0009] Based on the above technical solution, the pharmaceutically acceptable excipients further include diluents, fillers, binders, disintegrants, and lubricants.

[0010] Based on the above technical solution, the dosage form of the drug further includes tablets, capsules, granules, powders, oral liquids, syrups, suspensions, solutions, drops, and pills.

[0011] Based on the above technical solution, the route of administration of the drug is further specified as oral administration.

[0012] Based on the above technical solution, the dosage of the drug is further 50~200mg / kg.

[0013] Based on the above technical solution, the American ginseng glycoprotein extract can further inhibit the expression of NLRP3, Caspase-1, GSDMD, IL-18, and IL-1β proteins, effectively alleviating liver inflammation and pyroptosis.

[0014] Based on the above technical solution, the American ginseng glycoprotein extract can further reduce serum ALT, AST and TG levels in ALD mice, effectively reducing liver damage and liver lipid accumulation.

[0015] Based on the above technical solution, the American ginseng glycoprotein extract can further increase the abundance of Firmicutes in the intestine and reduce the abundance of Bacteroidetes and Proteus, effectively restoring the abundance of the core intestinal flora and improving the intestinal flora imbalance caused by ALD.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. TD can effectively inhibit NLRP3 protein expression and its downstream targets, thereby effectively alleviating liver inflammation and pyroptosis.

[0017] 2. TD can increase the abundance of Firmicutes in the gut and decrease the abundance of Bacteroidetes and Proteus, effectively restoring the abundance of the core gut microbiota and improving the gut microbiota imbalance caused by ALD.

[0018] 3. American ginseng glycoprotein extract has a multi-target protective effect against alcoholic liver disease. It can reduce liver tissue inflammation and oxidative stress damage caused by alcohol. The ingredients are mild and have a high safety profile, providing a natural, mild and effective protective pathway for alcoholic liver damage. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0020] Figure 1 The image shows the H&E staining results of mouse liver tissue pathological morphology in Example 1 of this application (×200).

[0021] Figure 2 The levels of NLRP3 signaling pathway-related proteins in mouse liver in Example 1 of this application (##P<0.01 vsNormal); P < 0.01 (v vs Ethanol).

[0022] Figure 3 The dilution curves (n=6) of each sample show the effect of TD on the gut microbiota of ALD mice in Example 1 of this application.

[0023] Figure 4 This is a Venn diagram showing the effect of TD on the gut microbiota of ALD mice in Example 1 of this paper.

[0024] Figure 5 This is an NMDS analysis diagram of the effect of TD on the gut microbiota of ALD mice in Example 1 of this application.

[0025] Figure 6 This is a PCoA analysis diagram showing the effect of TD on the gut microbiota of ALD mice in Example 1 of this application.

[0026] Figure 7 This is an analysis of the relative abundance of TD in the gut microbiota of ALD mice at the phylum (A) and genus (B) levels in Example 1 of this application. Detailed Implementation

[0027] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0028] The reagents used in this invention are: American ginseng protein extract TD, which uses American ginseng as the main raw material and natural eutectic solvent aqueous solution as the solvent. The extract is obtained by sequentially soaking and stirring, ultrasonic extraction and microwave extraction. The extract is then subjected to subsequent operations such as alcohol precipitation and removal of free protein to obtain American ginseng glycoprotein (provided by Professor Fang Lei's team at Jinan University).

[0029] Silymarin, Sigma-Aldrich; Lieber-DeCarli liquid feed (TP4060D), Nantong Trofi Feed Technology Co., Ltd.; Aspartate aminotransferase kit (AST, C010-2-1), alanine aminotransferase kit (ALT, C009-2-1), triglyceride kit (TG, A110-1-1), Nanjing Jiancheng Bioengineering Institute; NLRP3 antibody, GAPDH antibody, Proteintech Group; Caspase-1 antibody, IL-1β antibody, IL-18 antibody, Wanleibio; GSDMD antibody, Sangon Biotech.

[0030] The experimental animals used in this invention were SPF-grade C57BL / 6 male mice, 8-10 weeks old, weighing 22-24g, purchased from Liaoning Changsheng Biotechnology Co., Ltd., production license number: SCXK(Liaoning)2020-0001.

[0031] Example 1 1. Laboratory animals and grouping Male SPF-grade C57BL / 6 mice, aged 8-10 weeks and weighing 22-24g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., production license number: SCXK(Liaoning)2020-0001. The animal experiments were approved by the Ethics Committee of the Affiliated Zhongshan Hospital of Dalian University (No. DWLL2019060).

[0032] Mice were randomly divided into six groups of eight each to establish a mouse NIAAA model. The groups were: normal control group, alcohol group (Ethanol), low-dose TD group (Ethanol + TD 50 mg / kg), medium-dose group (Ethanol + TD 100 mg / kg), high-dose group (Ethanol + TD 200 mg / kg), and silymarin group (Ethanol + Silymarin 100 mg / kg).

[0033] Mice were acclimatized to a liquid diet for 5 days. On day 6, the control group received the control liquid diet, while the other groups received a 5% (v / v) alcohol liquid diet for 10 days. During the modeling period, the TD group and silymarin group were administered the corresponding dose of the drug by gavage, while the control group and alcohol group were administered an equal volume of physiological saline by gavage (0.1 ml / 10 g) for 10 consecutive days. On day 16, all mice except the control group were administered 5 g / kg of alcohol by gavage once, while the control group mice were administered maltodextrin with the same caloric value by gavage. Nine hours later, the mice were sacrificed, and blood was collected from the eyeballs, serum, liver, and intestinal contents.

[0034] 2. Serum biochemical index determination Serum ALT, AST, and TG levels were measured according to the kit instructions.

[0035] The test results are shown in Table 1. The results showed that alcohol intake aggravated hepatocellular damage, as evidenced by the significantly higher serum ALT, AST, and TG levels in the Ethanol group compared to the Normal group (P < 0.01). After administration of TD, serum ALT, AST, and TG levels significantly decreased. This indicates that TD effectively alleviated alcohol-induced liver damage.

[0036] Table 1. Serum ALT, AST, and TG levels in each group of mice ( x ± s, n = 6)

[0037] ##P < 0.01 vs Normal; P < 0.01 vs Ethanol 3. Liver pathological analysis Paraffin sections of mouse liver were routinely stained with H&E to observe pathological changes in the liver.

[0038] The results are as follows Figure 1 As shown, H&E staining results revealed that in the Ethanol group, liver parenchymal cells were damaged and fragmented, with abundant inflammatory cell infiltration around the central vein and lipid droplets of varying sizes forming a dense, net-like pattern in the cytoplasm. After TD intervention, hepatocyte damage was reduced, inflammatory cell infiltration decreased, and intracytoplasmic lipid droplets decreased. This indicates that TD can alleviate alcohol-induced liver morphological and structural damage and lipid accumulation.

[0039] 4. Protein Immunoblotting Experiment Liver tissue was added to RIPA lysis buffer containing protease inhibitors, incubated on ice for 30 minutes, and then centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was used for BCA protein quantification. The supernatant was added and boiled for denaturation. The protein samples were separated by SDS-PAGE electrophoresis and then wet-transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour, and then incubated overnight at 4°C with primary antibody. After washing with PBST, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 hour. The membrane was then visualized and imaged using ECL chemiluminescence to detect the expression of NLRP3, Caspase-1, GSDMD, IL-18, and IL-1β proteins. The results were standardized using GAPDH as an internal control.

[0040] The results are as follows Figure 2As shown, the results indicated that compared with the Normal group, alcohol intake significantly increased the protein expression of NLRP3, Caspase-1, GSDMD, IL-18, and IL-1β in the mouse liver, while under TD intervention, the expression of NLRP3 and its related proteins was significantly reduced (P < 0.01).

[0041] 5.16S rRNA sequencing analysis Intestinal contents of mice from each group were collected for microbial diversity analysis, and the results are as follows: Figures 3-7 As shown.

[0042] Shannon dilution curves show that ( Figure 3 As the number of samples and sequences increased, the curves for each group of mouse samples gradually flattened, indicating that the number of sequences had covered most of the bacterial species in the samples.

[0043] Venn graph analysis shows that ( Figure 4 There were 130 common OTUs among the six groups. The gut microbiota of the six groups of mice shared a common core microbiota in terms of species composition, while also having their own unique species, which laid the foundation for subsequent analysis of differences in microbiota structure.

[0044] β-diversity analysis showed that ( Figures 5-6 The Normal and Ethanol groups showed a clear trend of separation, suggesting that the gut microbiota composition of the Ethanol group mice was significantly altered compared to the Normal group. Under TD intervention, the TD group gradually approached the Normal group in a concentration-dependent manner.

[0045] Analysis of the phylum-level structure of the mouse gut microbiota revealed that ( Figure 7 (A) Compared with the control group, Ethanol decreased the abundance of Firmicutes and increased the abundance of Bacteroidetes and Proteobacteria in the mouse gut. Compared with the model group, administration of TD increased the abundance of Firmicutes and decreased the abundance of Bacteroidetes and Proteobacteria.

[0046] At the subordinate level ( Figure 7 B) Alcohol stimulation led to a decrease in the abundance of beneficial bacteria such as Erysipelothrix, Duroc, and Lactobacillus in the mouse gut, while the abundance of harmful bacteria such as Unclassified Styloides and Parabacterium increased. TD administration reversed this situation, indicating that TD showed a certain regulatory effect on the gut microbiota in the sequencing results.

[0047] Alcohol intake not only directly poisons the liver but also disrupts intestinal barrier function and permeability, as well as the balance of pathogenic microorganisms in the gut, leading to pathogen translocation to the liver and exacerbating liver damage. TD significantly reduced serum ALT, AST, and TG levels in ALD mice, effectively reducing liver damage and lipid accumulation. Alcohol activates NLRP3 expression, but under TD intervention, NLRP3 protein expression and its downstream targets were inhibited, indicating that TD can effectively alleviate liver inflammation and pyroptosis. Furthermore, TD increased the abundance of Firmicutes and decreased the abundance of Bacteroidetes and Proteus, effectively restoring the abundance of the core gut microbiota and improving the gut microbiota imbalance in ALD mice.

[0048] In summary, TD exerts a protective effect on ALD mice by inhibiting the NLRP3 inflammasome and improving gut microbiota, providing a reference for the treatment of ALD and the research and development of innovative drugs.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of American ginseng glycoprotein extract in the preparation of drugs for treating alcoholic liver disease.

2. The application according to claim 1, characterized in that, The American ginseng glycoprotein extract is obtained by extracting American ginseng.

3. The application according to claim 1, characterized in that, The drug comprises an effective amount of ginseng glycoprotein extract and pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, Pharmaceutically acceptable excipients include diluents, fillers, binders, disintegrants, and lubricants.

5. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, granules, powders, oral liquids, syrups, suspensions, solutions, droplets, and pills.

6. The application according to claim 1, characterized in that, The drug is administered orally.

7. The application according to claim 1, characterized in that, The dosage of the drug is 50~200mg / kg.

8. The application according to claim 1, characterized in that, The American ginseng glycoprotein extract can inhibit the expression of NLRP3, Caspase-1, GSDMD, IL-18, and IL-1β proteins, effectively alleviating liver inflammation and pyroptosis.

9. The application according to claim 1, characterized in that, The American ginseng glycoprotein extract can reduce serum ALT, AST and TG levels in ALD mice, effectively reducing liver damage and lipid accumulation in the liver.

10. The application according to claim 1, characterized in that, The American ginseng glycoprotein extract can increase the abundance of Firmicutes in the gut, reduce the abundance of Bacteroidetes and Proteus, effectively restore the abundance of the core gut microbiota, and improve the gut microbiota imbalance caused by ALD.

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