A composition for alleviating fatty liver and use thereof

Abstract

This invention provides a composition for alleviating fatty liver and its application, belonging to the field of pharmaceutical technology. The composition of this invention comprises the following components: dihydroberberine, neoruscosaponin, physalicylate, and trans-trimethoxyresveratrol, wherein the final concentrations of dihydroberberine, neoruscosaponin, physalicylate, and trans-trimethoxyresveratrol are all 5-15 μM. The composition of this invention can effectively alleviate hepatic fat accumulation, impaired liver function, and oxidative stress damage to the liver, and as a new product for the prevention and treatment of fatty liver, it has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a composition for relieving fatty liver and its application. Background Technology

[0002] miR-122 is a hepatocyte-specific microRNA that plays a crucial role in hepatic lipid metabolism. Studies have shown that miR-122 can prevent and treat fatty liver disease; however, gene therapy involving direct exogenous delivery of miR-122 is costly, inconvenient, and has uncertain safety profiles, making it relatively unsuitable for large-scale preventative treatment. Natural molecules, such as plant and animal extracts, offer advantages such as high safety, broad applicability, and cost-effectiveness. They can regulate hepatic miR-122 through natural active molecules to achieve disease prevention and treatment. Existing research only examines the regulatory effects of single or a limited number of natural molecules on miR-122 expression, lacking a comprehensive evaluation and screening of natural molecules, thus failing to determine the optimal combination of natural molecules for upregulating miR-122. Therefore, obtaining a combination of natural molecules that can alleviate fatty liver disease is imperative. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a composition for alleviating fatty liver and its application. The composition of this invention can effectively alleviate hepatic fat accumulation, impaired liver function, and oxidative stress damage to the liver, and as a new product for the prevention and control of fatty liver, it has good application prospects.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a composition for relieving fatty liver, the composition comprising the following components: dihydroberberine, neoruscogenin, hispidulin, and trans-trimethoxyresveratrol, wherein the final concentrations of dihydroberberine, neoruscogenin, hispidulin, and trans-trimethoxyresveratrol are all 5-15 μM.

[0006] Preferably, the dosage of the composition is 5-15 mg / kg.

[0007] Preferably, the composition further includes a pharmaceutically acceptable carrier.

[0008] This invention provides the use of the described composition in the preparation of a treatment for fatty liver.

[0009] The present invention also provides the use of the described composition in the preparation of agents for treating liver function impairment.

[0010] The present invention also provides the use of the described composition in the preparation of an agent for treating liver tissue inflammation.

[0011] Compared with existing technologies, this invention has the following beneficial effects: Utilizing a miR-122 reporter gene expression cell model, this invention achieved high-throughput screening of 2543 natural product molecules, discovering a batch of natural monomers with good regulatory effects. Orthogonal experiments revealed that the combination of four natural molecules—Dihydroberberine, Neoruscogenin, Hispidulin, and trans-Trimethoxyresveratrol—exhibited the best effect, overcoming the limited efficacy of single natural molecules while avoiding the increased costs and potential side effects associated with excessive combinations. Furthermore, animal models of fatty liver demonstrated that this combination of natural molecules effectively alleviates fatty liver disease, suggesting it as a novel method and product for the prevention and control of fatty liver, with promising application prospects. Attached Figure Description

[0012] Figure 1 To enable high-throughput screening of natural molecules that regulate miR-122 expression;

[0013] Figure 2 The expression of miR-122 in Huh7 cells after natural molecular intervention for 24 hours;

[0014] Figure 3 The effects of nine natural product molecules on cell activity;

[0015] Figure 4 Orthogonal experiments were conducted to regulate miR-122 expression by seven molecules.

[0016] Figure 5 To analyze the unimolecule effect by fitting the orthogonal experimental results using a linear regression model (lm function), where... This indicates that p < 0.05; This indicates that p < 0.001;

[0017] Figure 6 To analyze the main effects using analysis of variance (ANOVA), where... This indicates that p < 0.05;

[0018] Figure 7 To compare the effects of combining four natural molecules (4-NP) and seven natural molecules (7-NP) on the regulation of miR-122 expression, among which... This indicates that p < 0.001;

[0019] Figure 8 A flowchart for animal experiments;

[0020] Figure 9 The study aimed to investigate the effects of DNHT natural molecular combinations on fatty liver, where A represents the effect of DNHT on miR-122 expression in mouse liver; B represents the effect of DNHT on triglyceride (TG) content in liver; C represents the effect of DNHT on total cholesterol (TC) content in liver; and D represents the effect of DNHT on peritesticular fat weight and body weight ratio in mice.

[0021] Figure 10 The staining results of DNHT on pathological damage in liver tissue;

[0022] Figure 11 The effects of DNHT on the levels of LPO and MDA, indicators of lipid peroxidation damage in liver tissue, and serum ALT and AST levels are as follows: A represents the effect of DNHT on LPO, an indicator of lipid peroxidation damage in liver tissue; B represents the effect of DNHT on MDA, an indicator of lipid peroxidation damage in liver tissue; C represents the effect of DNHT on serum ALT levels; and D represents the effect of DNHT on serum AST levels. Detailed Implementation

[0023] This invention provides a composition (DNHT) for relieving fatty liver, the composition comprising the following components: dihydroberberine, neoruscogenin, hispidulin, and trans-trimethoxyresveratrol, wherein the final concentrations of dihydroberberine, neoruscogenin, hispidulin, and trans-trimethoxyresveratrol are all 5-15 μM, preferably 8-12 μM, and more preferably 10 μM.

[0024] In this invention, the dosage of the composition is 5-15 mg / kg, preferably 8-12 mg / kg, and more preferably 10 mg / kg. The composition is used orally or by injection, preferably by injection.

[0025] In this invention, the composition further includes a pharmaceutically acceptable carrier.

[0026] This invention provides the use of the described composition in the preparation of a treatment for fatty liver.

[0027] The present invention also provides the use of the described composition in the preparation of agents for treating liver function impairment.

[0028] The present invention also provides the use of the described composition in the preparation of an agent for treating liver tissue inflammation.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1: High-throughput screening of natural molecules regulating miR-122 expression

[0031] Each small molecule from a library of 2543 natural molecules (purchased from Selleck, catalog number L1400) was seeded with Huh7 cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) containing the miR-122 luciferase reporter gene in 96-well plates. After 24 hours of incubation at 37°C, the intracellular luciferase content was detected using a firefly luciferase assay kit (provided by Beyotime Biotechnology Co., Ltd., catalog number RG051M), reflecting the expression level of miR-122. The final concentration of each small molecule was 10 μM, and the number of Huh7 cells was 8000 per well. The luminescence intensity in each well was read using a microplate reader, and the fold change in luminescence intensity was calculated based on the blank control, thus enabling high-throughput detection of the regulatory effect of small molecules on miR-122 expression. Results are shown below. Figure 1 .

[0032] Depend on Figure 1 It was found that 2543 natural molecules were used to intervene in miR-122 luciferase reporter gene Huh7 cells at a concentration of 10 μM. After 24 hours, the intracellular luciferase content was detected by measuring the luminescence intensity. The change in luminescence intensity was obtained by comparing the cells with those without natural molecules. The top 20 natural molecules with regulatory effects were screened out as natural molecules that regulate miR-122 expression.

[0033] Example 2: Validation of molecules regulating miR-122 expression and determination of the optimal combination

[0034] The 20 natural molecules screened in Example 1 were further validated. Huh7 cells were seeded at 100,000 cells per well in 12-well plates. After 24 hours, the 20 screened natural small molecules were added to the cells at a final concentration of 10 μM. RNA was extracted from the cells 24 hours after intervention and reverse transcribed using the Takara RR037A reagent kit with miR-122 and U6. The reverse transcription system is shown in Table 1, and the reverse transcription primers are shown in Table 3.

[0035] Table 1 Reverse transcription reaction system

[0036]

[0037] After reverse transcription, the relative expression of miR-122 and U6 was detected by real-time quantitative PCR. The PCR reaction system is shown in Table 2, and the primers used are shown in Table 3.

[0038] Table 2 Real-time quantitative PCR reaction system

[0039]

[0040] Table 3 Primer sequences for reverse transcription and real-time quantitative PCR

[0041]

[0042] The regulatory effects of the top 20 natural product molecules selected from the high-throughput screening results on miR-122 were detected by real-time quantitative PCR. The results are shown in [Figure number missing]. Figure 2 .

[0043] Depend on Figure 2 It was found that 9 of the molecules upregulated miR-122, and the difference was statistically significant (p < 0.001).

[0044] The effects of nine natural product molecules on cell viability were detected by CCK8 assay. Results are shown below. Figure 3 .

[0045] Depend on Figure 3 It is known that Leptomycin B is highly cytotoxic, and Tedizolid Phosphate is not a common natural product molecule; therefore, these two candidate molecules were excluded. The seven natural product molecules—Dihydroberberine, Neoruscogenin, Diosmetin, 6-Hydroxyflavone (6-HF), Oroxylin A, Hispidulin, and trans-Trimethoxyresveratrol—had relatively little effect on Huh7 cell viability. Further orthogonal experiments were conducted to determine the optimal combination.

[0046] Orthogonal experiments were conducted on seven natural molecules that regulate miR-122 and have low cytotoxicity to explore different combinations of natural molecules. The orthogonal experimental design is shown in Table 4, where 1 represents no small molecule intervention and 2 represents the addition of a small molecule intervention at a final concentration of 10 μM. miR-122 expression was detected after 24 h, and the optimal combination was determined by orthogonal analysis. The results are shown in Table 4. Figures 4-7 .

[0047] Table 4 Orthogonal Design Table

[0048]

[0049] Depend on Figure 4 Orthogonal experiments revealed that the combination of four molecules—Dihydroberberine, Neoruscogenin, Hispidulin, and trans-Trimethoxyresveratrol—strongly regulated miR-122 expression. Figure 5 The results showed that, using a linear regression model (lm function) to fit the orthogonal experimental results and analyze the single-molecule effect, the combination of Dihydroberberine, Neoruscogenin, Hispidulin, and trans-Trimethoxyresveratrol significantly regulated miR-122 expression. Figure 6 The results showed that, using ANOVA (analysis of variance using the Aov function) to analyze the main effect, the combination of Dihydroberberine, Neoruscogenin, Hispidulin, and trans-Trimethoxyresveratrol significantly contributed to the main effect. Further comparisons were made of the regulatory effects of four natural molecules (Dihydroberberine, Neoruscogenin, Hispidulin, and trans-Trimethoxyresveratrol) (4-NP) and seven natural molecules (7-NP) on miR-122 expression. Figure 7 The combined effect of four natural molecules is equivalent to or even better than that of seven natural molecules. This demonstrates that using a combination of Dihydroberberine, Neoruscogenin, Hispidulin, and trans-Trimethoxyresveratrol can enhance the intervention effect of a single natural molecule while avoiding the increased costs and potential side effects associated with excessive combinations of natural molecules.

[0050] Example 3: The effect of DNHT in alleviating fatty liver

[0051] Eight-week-old C57 / BL6 mice (purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd.) were fed a Western diet (purchased from Shanghai Pluten Biotechnology Co., Ltd.) to establish a fatty liver animal model. The Western diet contained high fat (40% fat for energy), high fructose (20%), and high cholesterol (2%). Mice were first induced to have fatty liver by receiving the Western diet for 8 weeks, followed by 4 weeks of Western diet combined with a natural DNHT molecule combination. Figure 8 The intervention involved intraperitoneal injection of a natural DNHT molecular combination twice a week during week 9. The initial intervention dose was determined to be 10 mg / kg based on a 10 μM concentration used in cell experiments. The experiment concluded after 4 weeks of intervention, extending to a total feeding cycle of 12 weeks. Blood and liver tissue samples were collected from anesthetized mice to examine for fatty liver pathological changes and observe the effect of DNHT on fatty liver. Mice fed a standard diet (purchased from Shanghai Proton Biotechnology Co., Ltd.) were designated the CON group, mice fed a Western diet were designated the WD group, and mice fed a Western diet combined with DNHT were designated the DNHT group. Results are shown below. Figures 9-11 .

[0052] Depend on Figure 9 It can be seen that DNHT can significantly upregulate the expression level of miR-122 in the liver. Figure 10 It can be seen that Oil Red staining shows that DNHT reduces hepatic fat accumulation; Sirius Red staining shows that DNHT reduces hepatic collagen hyperplasia and fibrosis; and F4 / 80 immunohistochemical staining shows that DNHT reduces hepatic inflammatory response. Figure 11 It is known that DNHT can reduce the levels of LPO and MDA, indicators of lipid peroxidation damage in liver tissue, as well as the levels of ALT and AST in serum, thus alleviating liver function damage in a fatty liver model. Therefore, DNHT can significantly reduce fat accumulation, inhibit inflammation and fibrosis, and alleviate oxidative stress and liver function damage, thereby effectively alleviating fatty liver and related liver damage induced by a Western diet.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition for relieving fatty liver, characterized in that, The composition comprises the following components: dihydroberberine, neoruscosaponin, physalisin, and trans-trimethoxyresveratrol, wherein the final concentrations of dihydroberberine, neoruscosaponin, physalisin, and trans-trimethoxyresveratrol are all 5-15 μM. The composition functions to regulate miR-122 expression.

2. The composition according to claim 1, characterized in that, The dosage of the composition is 5-15 mg / kg.

3. The composition according to claim 1, characterized in that, The composition also includes a pharmaceutically acceptable carrier.

4. The use of the composition according to any one of claims 1 to 3 in the preparation of a treatment for fatty liver.

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