Application of inulin in preparation of medicine for treating fatty liver disease

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of inulin in preparation of a medicine for treating fatty liver diseases. The treatment effect of the inulin on the metabolism-related fatty liver disease is analyzed and proved, and a new idea is provided for research and development of MAFLD specific drugs in the future; meanwhile, a new treatment mechanism and molecular targets c-di-GMP and VAMP8 are excavated, it is found that improvement of recovery of the c-di-GMP activated VAMP8 mediated autophagy function is possibly a new target for treating MAFLD, and a new thought is provided for breaking through the problems that existing medicine treatment is lack of pertinence and poor in treatment effect in the future.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of inulin in the preparation of drugs for treating fatty liver disease. Background Technology

[0002] Metabolic fatty liver disease (MAFLD) is the most common chronic liver disease, with a prevalence of approximately 50.7% in overweight and obese adults. Without timely intervention, MAFLD can progress from simple steatosis to hepatitis, cirrhosis, and even liver cancer. During this process, damaged or dysfunctional organelles accumulate in cells, making autophagy crucial for processing these harmful cellular components. Increasing research recognizes a close correlation between autophagy dysfunction and the development of MAFLD, suggesting that targeting autophagy is a promising strategy for the prevention and treatment of MAFLD.

[0003] The gut is a complex organ with multiple functions. It not only plays a vital role in digestion and absorption but also serves as a habitat for a large number of microorganisms. These microorganisms participate in nutrient absorption, help maintain the intestinal barrier function to prevent pathogen invasion, improve the function of the intestinal immune system, and regulate metabolism, which is crucial for maintaining the body's energy balance. The gut is directly connected to the liver via the portal vein and can also directly regulate liver metabolism. Imbalances in the interaction between the gut and liver may be a significant cause of the development and progression of diseases such as MAFLD. A damaged intestinal barrier allows various exogenous pathogen-associated molecules to enter the liver, accelerating disease progression. Among these, cyclic diguanosine monophosphate (c-di-GMP) is a naturally occurring cyclic nucleotide produced by bacteria. Once in the body, it directly activates the innate immune effector STING and its downstream TBK1. However, the role of c-di-GMP in MAFLD remains unknown.

[0004] VAMP8 (Vesicle-Associated Membrane Protein 8) is a SNARE protein that is mainly involved in the transport and fusion of intracellular vesicles, playing a key role, especially in the endosome-lysosome pathway and autophagosome maturation. It mediates the fusion of autophagosomes and lysosomes by forming complexes with other SNARE proteins, thereby promoting the degradation of autophagy substrates. Activation of VAMP8 will inhibit the formation of SNARE complexes, thereby blocking autophagosome-lysosome fusion. Therefore, VAMP8 may be a key target for regulating liver autophagy dysfunction.

[0005] Inulin is a non-digestible dietary fiber found in the herbaceous plant Jerusalem artichoke. It can be fermented by beneficial bacteria in the gut. As a gut prebiotic, inulin can alleviate metabolic disorders by restoring the diversity of gut microbiota and plays an important role in metabolic diseases. However, current research on the mechanism of action of inulin is very limited. Summary of the Invention

[0006] The purpose of this invention is to provide the application of inulin in the preparation of medicaments for treating fatty liver disease, thereby addressing the problems mentioned in the background section.

[0007] The present invention is implemented in the application of inulin in the preparation of a drug for treating fatty liver disease.

[0008] Preferably, the fatty liver disease is caused by intestinal microbial c-di-GMP activating hepatic VAMP8, leading to autophagy dysfunction.

[0009] Preferably, the inulin improves hepatic lipid accumulation and abnormal liver function.

[0010] Preferably, the inulin enhances the intestinal barrier function.

[0011] Preferably, the inulin enhances liver autophagy function.

[0012] Preferably, the drug further includes pharmaceutically acceptable excipients or carriers.

[0013] Preferably, the dosage form of the drug is one of tablets, pills, or capsules.

[0014] The embodiments of this invention have confirmed the therapeutic effect of inulin on metabolic-associated fatty liver disease (MAFLD), providing new ideas for the future development of specific drugs for MAFLD. At the same time, new therapeutic mechanisms and molecular targets c-di-GMP and VAMP8 have been discovered. It has been found that improving the recovery of autophagy function mediated by c-di-GMP-activated VAMP8 may be a new target for the treatment of MAFLD, providing new ideas for overcoming the problems of lack of specificity and poor therapeutic effects of existing drug treatments. Attached Figure Description

[0015] Figure 1 The results of the degree of damage to the intestinal barrier and the accumulation of microbial c-di-GMP in MAFLD mice provided in Example 1 of the present invention; Figure 2 The results of immunofluorescence staining intensity of ZO-1 provided in Example 1 of this invention; Figure 3 The statistical analysis results of ZO-1 provided in Embodiment 1 of the present invention; Figure 4 The results of the effect of inulin on lipid accumulation in the liver of MAFLD mice provided in Example 2 of the present invention; Figure 5 The results of the effect of inulin on liver function improvement in MAFLD mice provided in Example 2 of the present invention; Figure 6 This is the result of the effect of inulin on improving intestinal barrier function in MAFLD mice provided in Example 3 of the present invention; Figure 7 The results of the animal experiment provided in Example 4 of this invention show the effect of inulin on the liver autophagy function of MAFLD mice. Figure 8 This is the result of the effect of c-di-GMP on hepatocytes provided in Example 4 of the present invention; Figure 9 The results of the cell experiment provided in Example 4 of this invention show the effect of inulin on the liver autophagy function of MAFLD mice. Figure 10 The results of transmission electron microscopy and IP experiments in the cell experiments provided in Example 4 of the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0018] Example 1: Determining the extent of intestinal barrier damage and the accumulation of microbial-derived c-di-GMP in MAFLD mice: (1) Establishment of experimental animal model: Eight-week-old male C57BL / 6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected and fed a high-fat diet (60 kcal% fat). They were also given a single dose of streptozotocin (STZ, Sigma, USA) via intraperitoneal injection. Seven days after STZ injection, fasting blood glucose was measured in the mice. Mice with fasting blood glucose ≥250 mg / dL were included in the disease group and continued to be fed a high-fat diet. Age-matched control mice were fed a normal diet (10 kcal% fat; both the high-fat diet and the normal diet were from Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd.) and were then given an intraperitoneal injection of 0.1 mol / L (pH 4.5) citrate buffer as an STZ control. (2) Experimental results: Using the cyclic diguanylic acid c-di-GMP detection kit, such as Figure 1As shown, a high-fat diet combined with streptozotocin resulted in the enrichment of microbial-derived cyclic dinucleotide c-di-GMP in the systemic circulation and liver of mice. Figure 1 (A and B); qPCR detection revealed that a high-fat diet combined with streptozotocin increased the 16S rDNA content in the systemic circulation and liver of mice. Figure 1 The results (C and D) suggest that microbial metabolites are enriched in MAFLD mice; Western blot experiments revealed a significant decrease in the expression of the intestinal tight junction proteins ZO-1 and Claudin1 in MAFLD mice. Figure 1 (E), ZO-1 ( Figure 1 (f) and Claudin1 ( Figure 1 There are significant differences between China and G; for example Figure 2 As shown, the immunofluorescence staining intensity of ZO-1 decreased, as... Figure 3 As shown, statistical analysis revealed significant differences, suggesting that the intestinal barrier of MAFLD mice was impaired.

[0019] Example 2: Clarifying the effect of inulin on lipid accumulation and liver function in MAFLD mice: (1) The method for establishing the experimental animal model is the same as in Example 1; (2) Grouping and intervention of experimental animals: After successful modeling, mice in the control group and the diabetic group were randomly divided into two groups: control group (Control, n=6), control + inulin intervention group (Inulin, n=7), non-alcoholic fatty liver disease group (MAFLD, n=6), and non-alcoholic fatty liver disease combined with inulin intervention group (MAFLD+Inulin, n=7). The weight of mice was measured regularly, and each intervention group was given 5 g / kg inulin according to the weight of each mouse (the inulin dosage was calculated according to the weight of the mouse, and the inulin was dissolved in drinking water for administration. The inulin was purchased from Cosucra Company in Belgium). (3) Experimental results: HE staining revealed that, for example Figure 4 As shown, a high-fat diet combined with streptozotocin resulted in numerous fat vacuoles and localized inflammatory cell infiltration in mouse hepatocytes, while inulin feeding reduced hepatocyte steatosis and inflammatory cell infiltration; Figure 5 As shown, the body weight and liver weight / body weight ratio of mice in the MAFLD group were significantly increased compared to the control group, while inulin feeding significantly reduced the body weight of mice in the MAFLD group. Figure 5 (B) and liver weight / body weight ratio ( Figure 5 (C) Detection using a kit revealed that the levels of TC and TG in the liver tissue of mice in the MAFLD group were significantly higher than those in the control group, while inulin treatment significantly reduced TC in the liver of mice. Figure 5 D) and TG content ( Figure 5Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are important indicators for detecting liver function. The serum AST and ALT levels in the MAFLD group mice were significantly higher than those in the control group mice, while inulin significantly reduced serum AST in MAFLD mice. Figure 5 (F) and ALT ( Figure 5 The levels of G (g) in inulin indicate that it alleviated liver function abnormalities in MAFLD mice.

[0020] Example 3: Clarifying the effect of inulin on intestinal barrier function in MAFLD mice: (1) The experimental animal model and grouping were the same as in Example 2; (2) Experimental results: such as Figure 6 As shown, Western blot experiments revealed that the expression of the intestinal tight junction protein Claudin1 in MAFLD mice was significantly reduced compared to the control group, while inulin restored the expression of intestinal Claudin1 protein. Figure 6 (A), and there are significant differences ( Figure 6 (B) Immunofluorescence staining of tight junction protein ZO-1 in paraffin sections of mouse intestines revealed that the fluorescence signal of ZO-1 in the intestines of MAFLD mice was significantly weaker than that in the control group. Figure 6 Inulin partially restored the fluorescence intensity of ZO-1, indicating that inulin enhanced the intestinal barrier in T2D mice. Figure 6 (D).

[0021] Example 4: Clarifying the effect of inulin on liver autophagy function in MAFLD mice: (1) The experimental animal model and grouping were the same as in Example 2; the cell experiments used the normal mouse hepatocyte AML12 cell line (purchased from Haixing Biotechnology, catalog number TCM-C709). (2) Experimental results: such as Figure 7 As shown, Western blot experiments revealed the expression of autophagy protein p62 in the liver of MAFLD mice. Figure 7 The ratio of LC3 (A) and LC3 (II) / LC3 (I) Figure 7 The expression of C was significantly increased in the inulin group, while it was significantly decreased in the MAFLD group compared to the MAFLD group, showing a clear difference. Figure 7 (B, D) indicates that inulin enhances autophagy function in the liver of MAFLD mice; p-STING (in the liver of MAFLD mice) Figure 7 (E), p-TBK1 ( Figure 7 The expression of G in the inulin group and its ratio to the total protein were significantly higher than those in the control group, while the expression of the above proteins was significantly lower in the inulin group. Figure 7The results (F and H) indicate that the STING-TBK1 signaling pathway is significantly activated in the liver of MAFLD mice, while inulin significantly inhibits the activity of the STING signaling pathway. The STING signaling pathway may be a key target for inulin to improve MAFLD. like Figure 8 As shown, elevated levels of the exogenous nucleotide molecule c-di-GMP were found in the liver of MAFLD mice. Kit testing revealed that inulin feeding significantly reduced c-di-GMP levels in the liver of MAFLD mice. Figure 8 (A); To further investigate the effects of c-di-GMP on hepatocytes in vitro, the AML12 hepatocyte cell line was selected. Western blot experiments showed that p62 protein expression significantly increased after c-di-GMP treatment, and p62 expression gradually increased with increasing c-di-GMP concentration. Figure 8 The results (B) indicate that p62 protein accumulation in hepatocytes is increased, and autophagy flux is inhibited. Furthermore, Western blot analysis revealed that c-di-GMP treatment significantly increased p-TBK1 expression and the p-TBK1 / TBK1 ratio. Compared to the control group, c-di-GMP treatment increased the p-STING / TBK1 ratio. When the c-di-GMP concentration reached 4 μg / ml, both STING and TBK1 were significantly activated. These results indicate that c-di-GMP significantly activates the STING-TBK1 signaling pathway in AML12 cells. Figure 9 In addition, Oil Red O staining revealed that low expression of STING effectively reversed c-di-GMP-induced lipid deposition in hepatocytes (C). Figure 9 (D) Western blot experiments revealed p62 protein accumulation and mTOR molecule activation ( Figure 9 (Middle E); the accumulation of autophagosomes was observed by transmission electron microscopy ( Figure 10 In addition, IP experiments revealed that c-di-GMP treatment significantly increased the phosphorylation level of VAMP8 in AML12 cells (F); Figure 10 (G) The above results indicate that the decrease in autophagy flux caused by c-di-GMP stimulation and the activation of the STING-TBK1-mTOR-VAMP8 signaling pathway may be the cause of lipid accumulation and decreased liver function in MAFLD mice, and are also one of the targets of inulin.

[0022] In summary, inulin can alleviate autophagy dysfunction and lipid accumulation in the liver of patients with fatty liver disease by targeting the reduction of c-di-GMP enrichment and the improvement of VAMP8 activation. This provides a new approach for the preparation of drugs to treat fatty liver disease. When using inulin to prepare drugs, other excipients or carriers can be added as needed, and the drugs can be prepared into tablets, pills, capsules, etc. as needed.

[0023] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of inulin in the preparation of drugs for treating fatty liver disease.

2. The application according to claim 1, characterized in that, The fatty liver disease is caused by intestinal microbial c-di-GMP activating hepatic VAMP8, leading to autophagy dysfunction.

3. The application according to claim 1, characterized in that, The inulin improves lipid accumulation in the liver and abnormal liver function.

4. The application according to claim 1, characterized in that, The inulin enhances the intestinal barrier function.

5. The application according to claim 1, characterized in that, The inulin enhances liver autophagy.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients or carriers.

7. The application according to claim 1, characterized in that, The dosage form of the drug is one of tablets, pills, or capsules.

Images