Application of combination of pioglitazone and ligustilide in prevention and treatment of metabolism-related fatty liver fibrosis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of combination of pioglitazone and ligustilide in prevention and treatment of metabolism-related fatty liver fibrosis. Specifically, the invention evaluates the protective effect of ligustilide alone in hepatic fibrosis caused by in-vivo and in-vitro high-fat and high-glucose stimulation for the first time. Meanwhile, in the evaluation process of improving related hepatic fibrosis injury by aiming at the pharmaceutical composition of pioglitazone and ligustilide, a high-fat and high-glucose model closely related to glucose and lipid metabolism disorder is innovatively constructed and selected. The combination of pioglitazone and ligustilide has a better curative effect than the single use of any one of pioglitazone and ligustilide, shows a strong synergistic effect in the aspects of lipid synthesis inhibition, lipid metabolism promotion, glycometabolism regulation and the like, provides a more effective and innovative prevention and treatment method for the clinical treatment of metabolism-related fatty liver fibrosis, and has a wide application prospect. Therefore, the method has important clinical significance and social benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of pioglitazone combined with ligustilide in the prevention and treatment of metabolic associated fatty liver fibrosis. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Liver fibrosis is a common pathological process in the progression of various chronic liver injuries to end-stage liver diseases. Its pathological manifestations are characterized by abnormal changes in the structure of hepatocytes (such as steatosis), intrahepatic metabolic disorders, diffuse inflammatory reactions, and explosive deposition of extracellular matrix (ECM). If not intervened in time, liver fibrosis may gradually progress to cirrhosis, liver failure, and more malignant hepatobiliary cell cancers. With the successful research, development, listing, and large-scale promotion of the hepatitis B vaccine, metabolic associated fatty liver fibrosis mainly related to glycolipid metabolism has become the most common type of liver fibrosis injury in clinical practice, which is also closely related to long-term high-calorie, high-fat, and high-sugar eating habits. Although many Western medicines have shown good anti-fibrotic activity in in vitro and in vivo experimental studies, currently the US FDA has not approved any specific drugs for the treatment of liver fibrosis. Therefore, there is an urgent need to develop effective prevention and treatment methods for metabolic associated fatty liver fibrosis.

[0004] The current drug strategy for the clinical treatment of metabolic associated fatty liver fibrosis focuses on improving insulin resistance and lipid metabolism disorders. Commonly used drugs include insulin sensitizers (such as thiazolidinediones), antioxidants (such as vitamin E), and lipid-lowering drugs (such as statins). Among them, pioglitazone, as a commonly used thiazolidinedione, activates peroxisome proliferators-activated receptor γ (PPARγ), enhances insulin sensitivity, reduces blood glucose and lipid levels, and has a positive therapeutic effect on fatty liver diseases related to metabolic syndrome. However, the clinical application of pioglitazone has many limitations. Clinical studies have shown that the long-term use of this drug may lead to weight gain, edema, reduced bone density, and increased cardiovascular risk. In addition, as a metabolic regulator, its anti-fibrotic effect is limited, and it can only delay the fibrosis process to a certain extent, and it is difficult to cover the multi-target pathological network of liver fibrosis, so it is difficult to fundamentally solve metabolic associated fatty liver fibrosis.

[0005] Meanwhile, traditional Chinese medicine has gradually become an important adjuvant or alternative therapy for fatty liver fibrosis in clinical practice due to its characteristics of multi-component and multi-target action. Currently, compound preparations such as Fuzheng Huayu Capsule and Qianggan Capsule have been applied to the clinical treatment of liver fibrosis. These Chinese patent medicines can simultaneously improve metabolic disorders and the fibrosis process through multi-pathway intervention, and this overall regulatory effect makes up for the limitations of single-target treatment with insulin sensitizers such as pioglitazone. At the same time, the preclinical studies of related drugs are not perfect, and the in vivo and in vitro disease models selected to simulate the clinical pathogenesis are not mature and may have certain side effects, resulting in the uncertainty of the anti-liver fibrosis efficacy of the active ingredients of traditional Chinese medicine and the difficulty in clinical promotion and application. Summary of the Invention

[0006] In view of the deficiencies in the above-mentioned existing technologies, through long-term technical and practical exploration, the inventors provide the application of pioglitazone combined with ligustilide in the prevention and treatment of metabolic-related fatty liver fibrosis. Specifically, the present invention first discovers and confirms through research that pioglitazone combined with ligustilide exhibits good synergistic effects in the prevention and treatment of diseases related to metabolic-related fatty liver fibrosis. Based on the above research results, the present invention is thus completed.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, there is provided the application of pioglitazone combined with ligustilide in any one or more of the following:

[0009] a) Preparing a product for inhibiting lipid synthesis;

[0010] b) Preparing a product for promoting lipid metabolism;

[0011] c) Preparing a product for regulating glucose metabolism;

[0012] d) Preparing a product for preventing and / or treating liver fibrosis.

[0013] In the present invention, the liver fibrosis further includes complications mediated by liver fibrosis. Further, the liver fibrosis is metabolic-related fatty liver fibrosis, and more specifically, it can be metabolic-related fatty liver fibrosis mediated by high fat and high sugar.

[0014] In the above application, the mass ratio of pioglitazone to ligustilide is 0.5 - 5:1, and further is 1:1. Based on the established in vivo and in vitro evaluation systems for fatty liver fibrosis, by using the composition of pioglitazone and ligustilide with the above mass ratio relationship, it is shown that the two exhibit good synergistic effects in the prevention and treatment of metabolic-related fatty liver fibrosis induced by high fat and high sugar.

[0015] Specifically, it at least shows the following effects: reducing hepatic steatosis induced by high-fat and high-sugar diet; improving the pathological condition of liver tissue, making the arrangement of hepatocytes tend to be normal and the number of lipid vacuoles decrease; reducing the degree of fatty degeneration and the number of lipid droplets; regulating the disordered process of glycolipid metabolism represented by key genes of glycolipid metabolism such as Fatty Acid Synthase (Fasn), Carnitine Palmitoyltransferase 1a (Cpt1a), Glucokinase (Gck), Tissue Inhibitor of Metalloprotease-1 (Timp1), collagen type I alpha 1chain Gene (Collagen1), 3-Hydroxy-3-Methylglutaryl-CoA Reductase (Hmgcr) and Glucose-6-phosphatase catalytic subunit 1 (G6pc1); inhibiting the process of liver fibrosis induced by high-fat and high-sugar diet.

[0016] In the second aspect of the present invention, there is provided a pharmaceutical composition, the active ingredient of which at least comprises pioglitazone and ligustilide.

[0017] When pioglitazone and ligustilide are used in combination, the mass ratio of the two is 0.5-5:1, and further preferably 1:1.

[0018] In the present invention, the pharmaceutical composition can be used for preventing and / or treating liver fibrosis.

[0019] In the third aspect of the present invention, there is provided a method for preventing and / or treating liver fibrosis, the method comprising administering the above-mentioned pharmaceutical composition to a subject.

[0020] Compared with the prior art solutions, the above one or more technical solutions have the following beneficial effects:

[0021] The above technical solution evaluated for the first time the protective effect of ligustilide alone against liver fibrosis induced by high-fat and high-sugar stimulation in vivo and in vitro. At the same time, in the evaluation process of the drug combination of pioglitazone and ligustilide for improving related liver fibrosis injury, the above technical solution did not select a single-factor model such as carbon tetrachloride or bile duct ligation that is divorced from the actual clinical pathological injury, but innovatively constructed and selected a high-fat and high-sugar model that is closely related to glycolipid metabolism disorders. It was found that the combined use of pioglitazone and ligustilide had significantly better efficacy than the use of either drug alone, showing a strong synergistic effect in inhibiting lipid synthesis, promoting lipid metabolism, regulating glucose metabolism, etc., providing a more effective and innovative prevention and treatment method for the clinical treatment of metabolic-related steatohepatitis fibrosis, and thus having important clinical significance and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 It is a graph showing the changes in the body weight and liver coefficient of mice in the examples of the present invention; compared with the control group, ***P < 0.01; compared with the high-fat and high-sugar group, ## P < 0.01, ### P < 0.001;

[0024] Figure 2 It is a morphological diagram of the liver of mice in the examples of the present invention;

[0025] Figure 3 It is a graph showing the results of H&E staining of the pathological structure changes of the liver of mice in the examples of the present invention;

[0026] Figure 4 It is the result of ADRP staining of hepatic steatosis in mice in the examples of the present invention;

[0027] Figure 5 It is a graph showing the results of Sirius Red staining of the pathological structure changes of the liver of mice in the examples of the present invention;

[0028] Figure 6 It is a graph showing the levels of Fasn, Cpt1a, Timp1, and Gck in the liver tissue of mice in the examples of the present invention; compared with the control group, *P < 0.5, **P < 0.1, ***P < 0.01; compared with the high-fat and high-sugar group, ## P < 0.01, ### P < 0.001;

[0029] Figure 7 It is a graph showing the results of oil red staining of AML12 cells in the examples of the present invention;

[0030] Figure 8 This is the graph of the change in glucose content in the AML12 cell culture medium in the embodiments of the present invention; compared with the control group, *P < 0.5; compared with the high-fat and high-sugar group, # P < 0.5.

[0031] Figure 9 This is the graph of the levels of Collagen1, Fasn, Hmgcr, and G6pc1 in AML12 cells in the embodiments of the present invention. Compared with the control group, *P < 0.5, **P < 0.1, ***P < 0.01; compared with the high-fat and high-sugar group, # P < 0.5, ## P < 0.1, ### P < 0.01. Detailed implementation manners

[0032] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations for the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0033] It should be noted that the terms used herein are only for describing the specific implementation manners and are not intended to limit the exemplary implementation manners according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0034] In a typical specific implementation manner of the present invention, the application of pioglitazone combined with ligustilide in any one or more of the following is provided:

[0035] a) Preparing a product for inhibiting lipid synthesis;

[0036] b) Preparing a product for promoting lipid metabolism;

[0037] c) Preparing a product for regulating glucose metabolism;

[0038] d) Preparing a product for preventing and / or treating liver fibrosis.

[0039] In the present invention, the product can be a drug or a test reagent for non-medical use. The test reagent can be used for basic research on liver fibrosis, especially metabolic-related steatohepatitis fibrosis, such as constructing relevant animal or cell models, so as to be used for mechanism research on metabolic-related steatohepatitis fibrosis, etc.

[0040] In the present invention, the liver fibrosis further includes complications mediated by liver fibrosis. Further, the liver fibrosis is metabolic associated fatty liver fibrosis, more specifically, metabolic associated fatty liver fibrosis mediated by high fat and high sugar, which is a pathological process of progressive liver injury accompanied by disorders of glucose and lipid metabolism.

[0041] In the application, the mass ratio of pioglitazone to ligustilide is 0.5 - 5:1, further 1:1. Based on the established in vivo and in vitro evaluation systems for fatty liver fibrosis, the combination of pioglitazone and ligustilide with the above mass ratio relationship shows good synergistic effects in preventing and treating metabolic associated fatty liver fibrosis induced by high fat and high sugar.

[0042] Specifically, it at least shows the following: alleviating liver steatosis induced by high fat and high sugar; improving the pathological condition of liver tissue, making the arrangement of hepatocytes tend to be normal and the number of lipid vacuoles decrease; reducing the degree of fatty degeneration and the number of lipid droplets; regulating the disordered process of glucose and lipid metabolism represented by key genes of glucose and lipid metabolism such as Fasn, Hmgcr, Cpt1a, Timp1, Gck, G6pc1, Collagen1; inhibiting the liver fibrosis process induced by high fat and high sugar.

[0043] In another specific embodiment of the present invention, a pharmaceutical composition is provided, and the active ingredients of the pharmaceutical composition at least include pioglitazone and ligustilide.

[0044] When pioglitazone and ligustilide are used in combination, the mass ratio of the two is 0.5 - 5:1, further 1:1.

[0045] In the present invention, the pharmaceutical composition can be used for preventing and / or treating liver fibrosis.

[0046] Further, the pharmaceutical composition further includes at least one non - pharmaceutical active ingredient.

[0047] The non - pharmaceutical active ingredients of the drug can be carriers, excipients, diluents, etc. commonly used in pharmacy. Moreover, according to the usual methods, it can be made into dosage forms such as powders, granules, suspensions, emulsions, syrups, sprays, etc. for oral administration, external use, suppositories and sterile injection solutions for use.

[0048] The non - pharmaceutical active ingredients such as carriers, excipients and diluents that can be included are well - known in the field, and those of ordinary skill in the art can determine that they meet clinical standards.

[0049] In yet another specific embodiment of the present invention, the carriers, excipients and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate and mineral oil, etc.

[0050] In yet another specific embodiment of the present invention, the drugs of the present invention can be administered into the body by known methods. For example, they can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as the target cells, the biological type or its tissue, the general condition of the subject to be treated, the administration route, the administration method, and so on.

[0051] In yet another specific embodiment of the present invention, the subjects to which the drugs are administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc.

[0052] In yet another specific embodiment of the present invention, there is provided a method for preventing and / or treating liver fibrosis, the method comprising administering an effective amount of the above-mentioned pharmaceutical composition to a subject.

[0053] The "subject" refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human.

[0054] The "effective amount" refers to the amount of the active compound or agent, including the compounds of the present invention, which can cause the biological or medical response of the tissue system, animal or human desired by the researcher, veterinarian, doctor or other medical personnel, which includes alleviating or partially alleviating the symptoms of the disease, syndrome, disorder or condition being treated. It must be recognized that the optimal dosage and interval of the active ingredient described in the present invention are determined by its nature and external conditions such as the form, route and site of administration and the specific mammal being treated, and this optimal dosage can be determined by conventional techniques. At the same time, it must also be recognized that the optimal course of treatment, that is, the daily dose of the compound within a rated time, can be determined by methods well known in the art.

[0055] The present invention will be further described below in conjunction with specific examples. The following examples are only for explaining the present invention and do not limit its content. Any simple modification, equivalent change and modification made to the embodiments based on the technical essence of the present invention all fall within the scope of the technical solution of the present invention.

[0056] Examples

[0057] I. Experimental Methods

[0058] 1. Construction of in vivo model

[0059] 1.1 Modeling and drug administration of experimental animals

[0060] (1) SPF-grade C57BL / 6 male mice, 8 weeks old, weighing 22 g - 24 g, were provided by Spf (Beijing) Biotechnology Co., Ltd.

[0061] The mice were randomly divided into 5 groups: control group, high-fat and high-sugar group, high-fat and high-sugar + ligustilide group (10 mg / kg), high-fat and high-sugar + pioglitazone group (10 mg / kg), high-fat and high-sugar + ligustilide + pioglitazone group (10 mg / kg + 10 mg / kg). After one week of adaptive feeding, the mice were modeled.

[0062] (2) Modeling and drug administration of experimental animals: Except for the control group, the remaining experimental mice were given a high-fat diet (42% Kcal fat and 0.2% cholesterol, TD88137) and high-sugar water (23.1 g / L d-fructose + 18.9 g / L d-glucose) and allowed to eat and drink freely for six weeks. After six weeks, each mouse was continuously treated by intragastric administration at a dose of 1 ml / 100 g body weight for ten weeks. The control group and the high-fat and high-sugar group were given an equal volume of normal saline.

[0063] 1.2 Sampling of experimental animals

[0064] The body weight of the mice was weighed weekly and the weight value was recorded. After anesthetizing the mice, blood was collected from the inferior vena cava. When enough blood was collected, the mice were sacrificed by cervical dislocation and then laparotomy was performed for sampling. The entire liver was cut off intact and placed in a culture dish. The liver morphology was photographed and the liver weight value was weighed and recorded. A piece of liver from the same location was placed in tissue fixative for subsequent morphological detection. The remaining liver tissue was placed in the corresponding cryotube, quickly frozen in liquid nitrogen, and then stored at -80 °C in the refrigerator for later use.

[0065] 1.3 Morphological detection of liver tissue

[0066] Hematoxylin-eosin (H&E) staining of mouse liver tissue was used for morphological detection of liver tissue.

[0067] 1.3.1 Experimental materials

[0068] Fixative: 4% paraformaldehyde, dehydrating agent: gradient ethanol (70%, 80%, 90%, 95%, 100%), clearing agent: xylene, embedding agent: paraffin, staining agents: hematoxylin stain and eosin stain (0.5% - 1% eosin aqueous solution), differentiating solution: 1% hydrochloric acid ethanol, bluing solution: Scott's bluing solution, mounting medium: neutral gum. Microtome, glass slides, coverslips, staining jars, oven, microscope.

[0069] 1.3.2 Experimental Methods

[0070] (1) Tissue fixation: Take a cube-shaped liver tissue block with a side length of 5 mm from the area near the portal vein of the large hepatic lobe of the mouse liver tissue, and immediately place it in 4% neutral paraformaldehyde for fixation for 24 - 48 h.

[0071] (2) Dehydration and clearing: After the liver tissue is fixed, it is dehydrated through a gradient of 70% ethanol (1 h) - 80% ethanol (1 h) - 90% ethanol (1 h) - 95% ethanol (1 h) - 100% ethanol (1 h) - 100% ethanol (1 h), and then cleared twice in xylene, 1 h each time.

[0072] (3) Paraffin embedding: Immerse the cleared tissue in melted paraffin (65 °C) twice, 1 h each time. Then, place the tissue in an embedding cassette and let it cool and solidify.

[0073] (4) Sectioning: Use a microtome to cut the paraffin block into 4.5-μm-thick sections. Float the sections on a 40 °C water bath to flatten them, pick them up onto glass slides, and dry them in an oven at 60 °C for 1 h.

[0074] (5) Deparaffinization and hydration: Place the tissue sections in xylene for deparaffinization, 3 times, 5 min each time, and then hydrate them through a gradient of 100% ethanol (5 min, three times) - 95% ethanol (5 min) - 80% ethanol (5 min) - 70% ethanol (5 min) - distilled water (5 min).

[0075] 100% ethanol (5 min, three times) - 95% ethanol (5 min) - 80% ethanol (5 min) - 70% ethanol (5 min) - distilled water (5 min) for gradient hydration.

[0076] (6) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 10 min, rinse with tap water for 1 min, and remove the excess staining solution.

[0077] (7) Differentiation and blueing: Immerse the sections in 1% hydrochloric acid ethanol for differentiation for 2 s (controlled under the microscope until the cell nuclei are clear and the background is colorless), and rinse with tap water for 1 min. Immerse the sections in Scott's blueing solution for 2 min, and rinse with tap water for 1 min.

[0078] (8) Eosin staining: Immerse the sections in eosin staining solution for 1 min, rinse with tap water for 30 s, and remove the excess staining solution.

[0079] (9) Dehydration and clearing: Pass the sections through a gradient of 70% ethanol (30 s) - 80% ethanol (30 s) - 90% ethanol (30 s) - 95% ethanol (1 min) - 100% ethanol (1 min) - 100% ethanol (1 min) - xylene (2 min) - xylene (2 min) for gradient dehydration and clearing.

[0080] (10) Mounting and observation: Add 1 - 2 drops of neutral balsam on the section, cover with a coverslip, and after air - drying at room temperature, observe the H&E - stained mouse liver tissue and acquire images using an Aperio Versa super - resolution imager.

[0081] The liver tissues were evaluated using the NAFLD Activity Score (NAS) scoring system proposed by the Non - alcoholic Steatohepatitis Clinical Research Network (NASH - CRN) of the United States in 2005. This scoring system evaluates the degrees of steatosis, lobular inflammation, and ballooning degeneration in the liver tissues of each group of mice. The heavier the degree, the higher the score. The criteria are shown in Table 1.

[0082] Table 1 NAS scoring criteria

[0083]

[0084]

[0085] 1.4 Morphological detection of liver tissue fat differentiation

[0086] The expression of adiposedifferentiation - related protein (ADRP) in mouse liver tissue was detected using immunofluorescence technology to evaluate the fat differentiation of liver tissue.

[0087] 1.4.1 Experimental materials

[0088] Gradient ethanol, xylene, sodium citrate antigen retrieval solution, BSA, goat serum, anti - ADRP antibody: rabbit anti - human ADRP monoclonal antibody, Alexa Fluor 594 - labeled fluorescent secondary antibody, DAPI.

[0089] 1.4.2 Experimental methods

[0090] (1) Deparaffinization and hydration: As described for H&E staining, after baking the liver tissue sections in an oven at 60 °C until the paraffin melts, they were successively deparaffinized in xylene (3 times, 5 minutes each time) and hydrated in gradient ethanol.

[0091] (2) Antigen retrieval: Immerse the sections in the pre - heated antigen retrieval solution (sodium citrate buffer), heat in a water bath to 85 °C, keep for 40 minutes, then cool to room temperature and rinse with distilled water for 5 minutes.

[0092] (3) Blocking: Prepare a blocking solution of 2.5% BSA PBS containing 0.2% Triton X - 100, and use this blocking solution to prepare a 10% goat serum solution. Drop it onto the sections and block at room temperature for 30 minutes to block non - specific binding sites.

[0093] (4) Primary antibody incubation: Add anti-ADRP antibody (1:200) diluted with blocking solution, place it in a wet box, and incubate overnight at 4°C in a refrigerator. After completion, rinse 3 times with 1X PBS, 3 minutes each time.

[0094] (5) Secondary antibody incubation: Add rabbit anti-conjugated with Alexa Fluor 594 fluorescence prepared with blocking solution to the tissue sections, incubate for 1 hour at room temperature in the dark. After incubation, wash the sections 3 times with PBS, 3 minutes each time.

[0095] (6) Mounting and image acquisition: Mount the sections with DAPI containing quencher, and acquire fluorescence images using a laser scanning confocal microscope (40×).

[0096] 1.5 Detection of liver tissue fibrosis

[0097] Use Sirius red staining to analyze the deposition of collagen fibers in liver tissue.

[0098] 1.5.1 Experimental materials

[0099] Xylene, gradient ethanol, hematoxylin staining solution, Sirius red staining solution, neutral resin.

[0100] 1.5.2 Experimental methods

[0101] (1) Deparaffinization and hydration: As described in H&E staining, after baking the liver tissue sections in an oven at 60°C until the paraffin melts, sequentially deparaffinize with xylene (3 times, 5 minutes each time) and hydrate with gradient ethanol (100% ethanol for 3 minutes - 95% ethanol for 3 minutes - 70% ethanol for 3 minutes), and finally place in distilled water for 5 minutes.

[0102] (2) Hematoxylin staining: Add hematoxylin staining solution, stain for 10 minutes at room temperature in the dark, wash with distilled water for 20 seconds to remove excess stain, and then wash with tap water for 5 minutes.

[0103] (3) Sirius red staining: Add Sirius red staining solution, stain for 20 minutes at room temperature in the dark, rinse with distilled water for 5 minutes to remove excess stain.

[0104] (4) Dehydration and mounting: Pass the sections sequentially through 70% ethanol for 1 minute - 95% ethanol for 1 minute - 100% ethanol for 3 minutes - xylene for 2 minutes - xylene for 2 minutes - xylene for 2 minutes, mount with neutral resin, air dry at room temperature, and then use an Aperio Versa super-resolution imager for staining observation and image acquisition.

[0105] 1.6 Expression of genes related to glycolipid metabolism in liver tissue

[0106] The mRNA levels of genes related to glycolipid metabolism, namely Fasn, Cpt1a, Timp1, and Gck, in mouse liver tissues were detected using reverse transcription-polymerase chain reaction (RT-PCR).

[0107] 1.6.1 Experimental materials

[0108] Trizol reagent, reverse transcription reagent, SYBR Green Master Mix, primers, chloroform, isopropanol, 75% ethanol (prepared with sterile and enzyme-free water), sterile and enzyme-free water, Nanodrop spectrophotometer, real-time fluorescence quantitative PCR instrument.

[0109] 1.6.2 Experimental methods

[0110] (1) RNA extraction: Add 100 mg of liver tissue to 700 μl of Trizol reagent, homogenize thoroughly, then add 200 μl of chloroform, vortex for ten times, 2 s each time, until completely mixed, and let it stand at room temperature for 10 min. Centrifuge at 12,000 g for 15 min at 4 °C, transfer the upper clear layer to another new EP tube, add an equal volume of isopropanol, invert 20 times up and down, and let it stand at room temperature for 15 min. Centrifuge at 12,000 g for 10 min at 4 °C, discard the supernatant, and a RNA precipitate can be seen. Add 900 μl of 75% alcohol (prepared with sterile and enzyme-free water), invert once up and down, centrifuge at 7500 g for 5 min at 4 °C, discard the supernatant, open the lid and let the EP tube lie on its side to dry the reagent, add sterile and enzyme-free water preheated at 45 °C to dissolve the RNA, and detect the RNA concentration using a ultra-micro ultraviolet-visible spectrophotometer (NanoDrop One).

[0111] (2) Reverse transcription: Perform reverse transcription according to the instructions of the reverse transcription kit. The total amount of RNA is 1 μg, and the reaction system is 20 μl. Calculate the required amount of RNA and the volume of sterile and enzyme-free water according to the RNA concentration. Sequentially add sterile and enzyme-free water, RNA, and 4 μl of 4×gDNAwiper mix to a 200 μl EP tube, react at 42 °C for 2 min; then add 4 μl of 5×HisScript III qRTSuper mix, react at 37 °C for 15 min, and finally maintain at 85 °C for the last 5 s.

[0112] (3) cDNA amplification and quantification: Dilute the cDNA mother liquor obtained by reverse transcription to 2 ng / μl. Prepare the reaction system for the corresponding primers according to the instructions of the amplification kit: 5 μl of cDNA working solution + 15 μl of MIX (10 μl of AceQ Universal SYBR qPCR Master Mix + 4.6 μl of enzyme-free and sterile water + 0.4 μl of primer). Use Hprt1 as the internal reference for detection. Set the amplification program in a real-time fluorescence quantitative PCR instrument: React at 95°C for 10 min, followed by 39 cycles of 95°C for 15 s and 60°C for 30 s. Read and record the copy number. Calculate the relative fold change in the mRNA expression level of the target gene: Relative mRNA expression level of the sample = Power[2, (Copy number of the target gene in the sample - Copy number of the Hprt1 gene in the sample) / (Copy number of the target gene in the control group - Copy number of the Hprt1 gene in the control group)].

[0113] 2. Construction of in vitro model

[0114] 2.1 Cell modeling and drug administration

[0115] AML12 (normal mouse hepatocytes) were cultured in DMEM medium containing 10% fetal bovine serum. When the cell density reached 90%, the cells were digested and passaged. The cells digested with trypsin were seeded in a six-well plate at a density of 500,000 cells per well. The experimental groups were: control group, high-fat and high-sugar group, high-fat and high-sugar + ligustilide group (20 μM), high-fat and high-sugar + pioglitazone group (10 μM), high-fat and high-sugar + ligustilide + pioglitazone group (20 μM + 10 μM). The next day, when the cell density reached 70%, the medium was replaced with DMEM medium containing 1% fetal bovine serum, and the cells were continued to be cultured in a cell incubator for 1 h. Subsequently, except for the control group, the remaining groups were stimulated with high-fat and high-sugar (palmitic acid + glucose, final concentration of 300 μM + 10 mM), and the corresponding concentrations of drugs. After 24 h, samples were collected according to different experimental purposes.

[0116] 2.2 Oil red O staining of AML12 cells

[0117] 2.2.1 Experimental materials

[0118] Isopropanol, oil red O staining solution (weigh 0.5 g of oil red O powder and dissolve it in 100 ml of isopropanol to obtain the oil red O stock solution. Then mix the oil red O stock solution with distilled water at a ratio of 3:2 to obtain the oil red O working solution. Let it stand for 10 min and then filter. Store it in the dark at 4°C), 4% paraformaldehyde fixative, inverted fluorescence microscope.

[0119] 2.2.2 Experimental methods

[0120] (1) Cell fixation: After the cell treatment was completed, the culture medium was discarded, and the cells were gently washed twice with PBS. 4% paraformaldehyde fixative was added, and the cells were fixed at room temperature for 30 min. Then the cells were washed twice with PBS, 3 min each time.

[0121] (2) Oil Red O staining: After cell fixation, the PBS was discarded, and Oil Red O working solution was added. The cells were stained at room temperature in the dark for 30 min. The staining solution was aspirated, and the cells were quickly rinsed with 60% isopropanol (5 - 10 s) to remove the excess staining solution. Subsequently, the cells were washed twice with PBS, 3 min each time. The staining results were observed with an inverted fluorescence microscope and images were acquired.

[0122] 2.3 Detection of glucose content in AML12 cell culture medium (glucose oxidase method)

[0123] 2.3.1 Experimental materials

[0124] Phosphate buffer, 3,5 - dichloro - 2 - hydroxybenzenesulfonate (DHBS), 4 - aminoantipyrine, glucose oxidase, magnesium chloride, peroxidase, glucose standard (5.55 mmol / L), distilled water, microplate reader, incubator.

[0125] 2.3.2 Experimental methods

[0126] (1) Sample treatment: The cell culture was taken out of the incubator, and the supernatant was collected. The cells and debris were removed by centrifugation at 1500 rpm for 10 min, and the supernatant was taken for subsequent detection.

[0127] (2) Preparation of glucose oxidase reagent: Using distilled water as the solvent, it was prepared according to the corresponding concentrations of phosphate buffer (100 mmol / L), 3,5 - dichloro - 2 - hydroxybenzenesulfonate (DHBS) (2 mmol / L), 4 - aminoantipyrine (1 mmol / L), glucose oxidase (10 kU / L), magnesium chloride (3.5 mmol / L), peroxidase (8 kU / L), and stored at 4℃ in the dark.

[0128] (3) Standard curve preparation and sample detection: 10 μL of distilled water (blank well), 10 μL of standard (standard well), and the supernatant of cell culture medium (sample well) were taken into a 96 - well plate. 250 μL of glucose oxidase reagent was added to each well, and the plate was gently shaken and incubated in a 37℃ incubator for 10 min. After incubation, the absorbance value of each well was measured at a wavelength of 505 nm with a microplate reader and recorded as A.

[0129] (4) Content calculation: Glucose content (mmol / L) = [(A measurement - A blank) / (A standard - A blank)] * 5.55.

[0130] 2.4 Gene expression related to glycolipid metabolism in AML12 cells

[0131] As described in 1.6, RT-PCR technology was used to detect the mRNA levels of Fasn, Hmgcr, Collagen1, and G6pc1 in AML12 cells.

[0132] II. Experimental results

[0133] 1. Changes in mouse body weight and liver coefficient

[0134] As Figure 1 shown, the experimental results show that: The body weight of the mice in the control group increased steadily during the 16-week feeding period, and the increase in body weight was within the normal range, with a relatively gentle growth curve. The body weight of the mice in the high-fat and high-sugar model group increased rapidly, indicating that the high-fat and high-sugar diet led to a significant increase in the body weight of the mice. After treatment with ligustilide, pioglitazone, and their drug combination, the growth rate of the mice's body weight decreased significantly. At the same time, compared with the control group, the liver coefficient of the mice in the high-fat and high-sugar model group also increased significantly, while ligustilide and their drug combination decreased the liver coefficient of the mice. Among them, the combination of pioglitazone and ligustilide had the most significant reducing effect, indicating that it alleviated the liver injury induced by high-fat and high-sugar.

[0135] 2. Changes in the liver morphology of mice

[0136] As Figure 2 shown, the experimental results show that: Compared with the control group, the livers of the mice in the high-fat and high-sugar group showed obvious pathological characteristics, with an enlarged liver volume, grayish-white color, round and blunt edges, and a fatty luster on the surface, suggesting obvious fatty degeneration of the liver due to excessive lipid accumulation. In addition, slight unevenness or punctate congestion could be seen, which was related to the tissue structure damage caused by lipotoxicity injury and inflammatory reaction. After treatment with ligustilide, pioglitazone, and their drug combination, the morphology of the mice's livers was significantly improved, the liver volume gradually returned to normal, the volume was smaller than that of the high-fat and high-sugar group, the color was ruddy, and the boundary was clear, indicating that these drugs could effectively alleviate the liver fat lesions induced by high-fat and high-sugar, and the combination of pioglitazone and ligustilide had the most significant improvement effect, and the liver morphology was close to that of the control group.

[0137] 3. Changes in the pathological structure of the mouse liver

[0138] As Figure 3As shown, the experimental results indicate that: in the high-fat and high-sugar group, the hepatocytes in the mouse liver tissue are arranged disorderly, the cell morphology is abnormal, and there are obvious fat vacuoles in the cytoplasm. A large number of closely arranged lipid droplets can be seen, indicating that obvious pathological features of fatty liver have occurred in mice induced by high-fat and high-sugar. After treatment with ligustilide, pioglitazone, and their drug combination, the pathological condition of the liver tissue has been significantly improved. The arrangement of hepatocytes tends to be normal, the number of fat vacuoles decreases, and the effect of the combination of pioglitazone and ligustilide is more significant, and the liver tissue structure is closer to that of the control group. The NAS score is shown in Table 2. The NAS score of the liver H&E of mice in the high-fat and high-sugar group is significantly increased, reaching 7.00 ± 0.63. Ligustilide (4.50 ± 1.05), pioglitazone (5.33 ± 1.37), and their combination (2.17 ± 1.17) all significantly reduce the NAS score, and the degree of reduction of the NAS score by the combination of ligustilide and pioglitazone is greater than that of single drug use and their sum, thus indicating that they produce a synergistic effect.

[0139] Table 2 NAS scores of the livers of mice in each group

[0140]

[0141] 4. Fatty change in mouse liver

[0142] As Figure 4 shown, compared with the control group, the accumulation of lipid droplets in the liver tissue of mice in the high-fat and high-sugar group is significantly increased, showing a strong green fluorescence signal, and the positive staining area reaches 34.01 ± 3.81%, showing a clustered distribution, indicating that high-fat and high-sugar treatment has led to a significant increase in fatty change in the mouse liver. After treatment with ligustilide (21.56 ± 2.12%), pioglitazone (23.05 ± 1.86%), and their drug combination (9.88 ± 1.95%), the degree of fatty change in the mouse liver is effectively reduced, the positive fluorescence staining area is significantly reduced, and the number of lipid droplets decreases. As shown in Table 3, compared with the high-sugar and high-fat group, the fluorescence intensity of the ligustilide single drug group is reduced by 36.61%, the fluorescence intensity of the pioglitazone single drug group is reduced by 32.20%, and the combination of the two reduces by 70.95%, with a more significant effect and returning to near the normal level.

[0143] Table 3 Statistics of ADRP staining in the liver tissues of mice in each group

[0144]

[0145]

[0146] 5. Changes in the pathological structure of mouse liver

[0147] As Figure 5As shown, the experimental results indicate that in the high-fat and high-sugar model group, Sirius red staining in the liver tissue of mice showed a significant increase in red collagen fiber deposition, mainly distributed around the hepatic sinusoids and portal areas, indicating that a high-fat and high-sugar diet induced a significant increase in the extracellular matrix and the occurrence of fibrosis in the liver. After treatment with ligustilide, pioglitazone, and their drug combination, the deposition of collagen fibers in the livers of mice decreased, and the combination of pioglitazone and ligustilide had a more significant effect, with the liver tissue structure being closer to that of the normal group. This indicates that ligustilide and pioglitazone can effectively inhibit the process of liver fibrosis induced by high fat and high sugar, improve the pathological state of the liver, and the combined use of drugs has a stronger anti-fibrotic effect. The specific statistical results of the positive staining area are shown in Table 4. Compared with the high-sugar and high-fat group, the area of collagen fiber deposition in the ligustilide single-drug group decreased by 43.18%, the positive area in the pioglitazone single-drug group decreased by 32.54%, and the combination of the two decreased by 79.47%, with a more significant effect and a recovery close to the normal level.

[0148] Table 4 Statistical results of Sirius Red staining in liver tissues of mice in each group

[0149]

[0150] 6. Changes in the levels of Fasn, Cpt1a, Gck, and Timp1 in mouse liver tissue

[0151] As Figure 6 shown, the experimental results indicate that compared with the control group, the expressions of fatty acid synthase-related gene Fasn, glucose metabolism regulatory gene Gck, and collagen degradation-related gene Timp1 in the liver tissue of mice in the high-fat and high-sugar group were significantly up-regulated, while the expression of carnitine palmitoyltransferase gene Cpt1a was significantly down-regulated. This indicates that high-fat and high-sugar stimulation led to glycolipid metabolism disorders and extracellular matrix deposition in the livers of mice. Ligustilide, pioglitazone, and their drug combination can all down-regulate the expression levels of Fasn, Gck, and Timp1, and up-regulate the expression of Cpt1a, and the regulatory effect of the combination of pioglitazone and ligustilide is more significant. This shows that both ligustilide and pioglitazone can improve the glycolipid metabolism disorders in the liver induced by high fat and high sugar by inhibiting lipid synthesis, promoting lipid metabolism, and regulating glucose metabolism, and the combination of pioglitazone and ligustilide has a better synergistic effect.

[0152] 7. Oil red staining results of AML12 cells

[0153] As Figure 7As shown in the figure, the experimental results show that: compared with the control group, a large number of red lipid droplets can be observed to aggregate in the cytoplasm in the high-fat and high-sugar model group, indicating a significant increase in intracellular lipid deposition, reflecting the disorder of lipid metabolism induced by high-fat and high-sugar in AML12 cells. After treatment with ligustilide, pioglitazone and their drug combination, the number and volume of red lipid droplets in the cells decreased significantly, and lipid deposition was significantly alleviated. Among them, the combination of pioglitazone and ligustilide had a more prominent effect. The specific statistical results of the positive staining area are shown in Table 5. Compared with the high-sugar and high-fat group, the deposition area of collagen fibers in the ligustilide single-drug group decreased by 33.17%, the positive area in the pioglitazone single-drug group decreased by 36.26%, and the combination of the two decreased by 79.82%, with a more significant effect, returning to near the normal level.

[0154] Table 5 Statistical results of the positive area of Oil Red staining of AML12 cells in each group

[0155]

[0156] 8. Changes in glucose content in the culture medium of AML12 cells

[0157] As Figure 8 shown in the figure, the experimental results show that: compared with the control group, the glucose content in the culture medium of AML12 cells in the high-fat and high-sugar group increased significantly, indicating that AML12 cells had glucose metabolism disorder, and the high-sugar and high-fat environment led to glucose uptake disorder and insulin resistance in AML12 cells. After treatment with ligustilide, pioglitazone and their drug combination, the glucose content in the culture medium could be reduced, effectively improving the abnormal glucose metabolism of cells. Among them, the combination of pioglitazone and ligustilide had a better effect.

[0158] 9. Changes in the levels of Collagen1, Fasn, Hmgcr, and G6pc1 in AML12 cells

[0159] As Figure 9 shown in the figure, the experimental results show that: compared with the control group, the high-fat and high-sugar model significantly up-regulated Fasn, the cholesterol synthesis-related gene Hmgcr, and the glucose metabolism gene G6pc1 in AML12 cells, and at the same time, the expression of the related collagen gene Collagen1 was also up-regulated. It shows that high-fat and high-sugar stimulation leads to glycolipid metabolism disorder in AML12 cells and causes an increase in the extracellular matrix. While ligustilide, pioglitazone and their drug combination can all down-regulate the expression of these key glycolipid metabolism genes, and the down-regulation effect of the combination of pioglitazone and ligustilide is more significant.

[0160] In summary, through the established in vivo and in vitro evaluation systems for fatty liver fibrosis, the present invention has comprehensively studied the pharmaceutical composition, and confirmed that it exhibits more significant therapeutic effects compared to the existing single-drug therapies, thus providing a new solution for the prevention and treatment of metabolic-related fatty liver fibrosis.

[0161] The foregoing are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. Use of pioglitazone combined with ligustilide in any one or more of the following: a) preparing products that inhibit lipid synthesis; b) preparing products that promote lipid metabolism; c) preparing products for regulating sugar metabolism; d) preparing products for preventing and / or treating liver fibrosis.

2. The use according to claim 1, characterized in that The product is a test reagent for pharmaceutical or non-medical use.

3. The use according to claim 1, characterized in that The liver fibrosis also includes complications mediated by liver fibrosis. Furthermore, the liver fibrosis is metabolism-related fatty liver fibrosis, more specifically metabolism-related fatty liver fibrosis mediated by high fat and high sugar.

4. The use according to claim 1, characterized in that: The mass ratio of pioglitazone to ligustilide is 0.5-5:1, further 1:

1.

5. The use according to claim 1, characterized in that: The treatment of liver fibrosis specifically manifests at least as follows: alleviating liver fatty lesions induced by high fat and high sugar; improving the pathological condition of liver tissue, so that the arrangement of liver cells tends to be normal and the number of fat vacuoles is reduced; the degree of fatty degeneration is reduced and the number of lipid droplets is reduced; regulating the disordered process of glucose and lipid metabolism represented by the key genes of glucose and lipid metabolism Fasn, Hmgcr, Cpt1a, Timp1, Gck, G6pc1, and Collagen1; and inhibiting the process of liver fibrosis induced by high fat and high sugar.

6. A pharmaceutical composition, characterized in that The active ingredients of the pharmaceutical composition at least include pioglitazone and ligustilide.

7. The pharmaceutical composition according to claim 6, characterized in that The mass ratio of pioglitazone to ligustilide is 0.5-5:1, further 1:

1.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is used for preventing and / or treating liver fibrosis.

9. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further comprises at least one non-pharmaceutical active ingredient; further, the non-pharmaceutical active ingredient is a carrier, excipient or diluent commonly used in pharmacy.

10. The pharmaceutical composition according to claim 6, characterized in that The subjects of drug administration are humans and non-human mammals.

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