Application of pioglitazone combined with ligustrazine lactone in prevention and treatment of metabolic related fatty liver fibrosis

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of pioglitazone combined with ligustilide in prevention and treatment of metabolic related fatty liver fibrosis. Specifically, the application first evaluates the protective effect of ligustilide alone in the liver fibrosis caused by high-fat and high-sugar stimulation in vivo and in vitro. Meanwhile, in the evaluation process of improving related liver fibrosis damage of the pioglitazone and ligustilide drug combination, the high-fat and high-sugar model closely related to glucose and lipid metabolism disorder is innovatively constructed and selected. It is found that the curative effect of the combination of pioglitazone and ligustilide is significantly better than that of any one of the drugs used alone, and the combination exhibits a strong synergistic effect in inhibiting lipid synthesis, promoting lipid metabolism, regulating glucose metabolism and the like, so that a more effective and innovative prevention and treatment method is provided for clinical treatment of metabolic related fatty liver fibrosis, and therefore has important clinical significance and social benefits.

Description

TECHNICAL FIELD

[0001] The present application 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-related fatty liver fibrosis. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] Liver fibrosis is a common pathological process of various chronic liver damage to end-stage liver disease, and its pathological manifestations are characterized by abnormal changes in liver cell structure (such as fatty degeneration), metabolic disorders in the liver, diffuse inflammatory response, and explosive extracellular matrix (ECM) deposition. If not intervened in time, liver fibrosis may gradually progress to cirrhosis, liver failure, and more malignant hepatobiliary cancer. With the successful development and large-scale promotion of hepatitis B vaccine, metabolic-related fatty liver fibrosis mainly caused by glucose and lipid metabolism has become the most common type of liver fibrosis damage in clinical practice, which is closely related to long-term high-calorie, high-fat, and high-sugar dietary habits. Although many western medicines have shown good anti-fibrosis activity in in vitro and in vivo experimental studies, there is currently no specific drug approved by the US FDA for the treatment of liver fibrosis. Therefore, it is urgent to develop an effective prevention and treatment method for metabolic-related fatty liver fibrosis.

[0004] The current drug strategy for the clinical treatment of metabolic-related fatty liver fibrosis focuses on improving insulin resistance and lipid metabolism disorders. Common ones include insulin sensitizers (such as thiazolidinedione drugs), antioxidants (such as vitamin E), and lipid-lowering drugs (such as statins). Among them, pioglitazone, as a commonly used thiazolidinedione drug, can enhance insulin sensitivity, reduce blood glucose and lipid levels, and has a positive therapeutic effect on fatty liver disease related to metabolic syndrome by activating peroxisome proliferator-activated receptor gamma (PPARγ). However, there are many limitations in the clinical application of pioglitazone. Clinical studies have shown that long-term use of the drug can lead to weight gain, edema, reduced bone density, and increased cardiovascular risk. In addition, as a metabolic regulator, its anti-fibrosis effect is limited, and it can only delay the progression of fibrosis to a certain extent, and it is difficult to cover the multi-target pathological network of liver fibrosis, and it is difficult to fundamentally solve metabolic-related fatty liver fibrosis.

[0005] Meanwhile, traditional Chinese medicine, with its multi-component and multi-target characteristics, has gradually become an important auxiliary or alternative therapy for clinical fatty liver fibrosis. At present, Fuzheng Huayu Capsules, Qianggan Capsules and other compound preparations have been applied to the clinical treatment of liver fibrosis. These Chinese patent medicines can improve metabolic disorders and fibrosis progression through multi-channel intervention, and this overall regulation can compensate for the limitations of single-target therapy of insulin sensitizers such as pioglitazone. At the same time, the preclinical research of related drugs is not perfect, and the in vitro and in vivo disease models simulating the clinical pathogenesis are not mature and may have certain side effects, leading to uncertainty of the anti-liver fibrosis effect of active ingredients of traditional Chinese medicine and difficulties in clinical application. SUMMARY

[0006] In view of the deficiencies in the prior art, the inventors have, through long-term technical and practical exploration, provided the application of pioglitazone combined with ligustilide in the prevention and treatment of metabolic-related fatty liver fibrosis. Specifically, the inventors have for the first time discovered and confirmed that pioglitazone combined with ligustilide exhibits a good synergistic effect in the prevention and treatment of metabolic-related fatty liver fibrosis-related diseases. Based on the above research results, the present application is completed.

[0007] To achieve the above technical purposes, the present application adopts the following technical solutions:

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

[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 application, the liver fibrosis also includes complications mediated by liver fibrosis, and further, the liver fibrosis is metabolic-related fatty liver fibrosis, and more specifically, it can be metabolic-related fatty liver fibrosis induced by high-fat and high-sugar.

[0014] In the application, the mass ratio of pioglitazone to ligustilide is 0.5-5:1, and further 1:1. Based on the constructed in vivo and in vitro fatty liver fibrosis evaluation system, the combination of pioglitazone and ligustilide in the above mass ratio shows a good synergistic effect in the prevention and treatment of metabolic-related fatty liver fibrosis induced by high-fat and high-sugar.

[0015] Specifically, at least the following aspects are embodied: alleviating high-fat high-sugar induced liver fatty lesions; improving the pathological condition of liver tissue, so that the arrangement of hepatocytes 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 glucose and lipid metabolism disorder process represented by key genes of glucose and lipid metabolism, such as fatty acid synthase (Fasn), carnitine palmitoyltransferase 1a (Cpt1a), glucokinase (Gck), tissue inhibitor of metalloprotease-1 (Timp1), collagen type I alpha 1 chain gene (Collagen1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), and glucose-6-phosphatase catalytic subunit 1 (G6pc1); inhibiting the process of high-fat high-sugar induced liver fibrosis.

[0016] In a second aspect of the present application, a pharmaceutical composition is provided, wherein the active ingredients of the pharmaceutical composition at least include pioglitazone and ligustilide.

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

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

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

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

[0021] The above technical scheme firstly evaluates the protective effect of ligustilide alone in the liver fibrosis caused by high-fat and high-sugar stimulation in vivo and in vitro. Meanwhile, in the evaluation of the improvement of the related liver fibrosis damage by the drug combination of pioglitazone and ligustilide, the above technical scheme does not use the single factor model such as carbon tetrachloride and bile duct ligation which is deviated from the clinical actual pathological damage, but innovatively constructs and selects the high-fat and high-sugar model closely related to the glucose and lipid metabolism disorder. It is found that the curative effect of the combination of pioglitazone and ligustilide is significantly better than that of any one of the drugs used alone, showing a strong synergistic effect in inhibiting lipid synthesis, promoting lipid metabolism, regulating glucose metabolism and the like, and providing a more effective and innovative prevention and treatment method for the clinical treatment of metabolic related fatty liver fibrosis, and therefore having important clinical significance and social benefits. BRIEF DESCRIPTION OF DRAWINGS

[0022] The drawings accompanying the specification of this application form a part thereof, serve to provide further understanding of the application, and together with the description of the exemplary embodiments of the application given below, make an explanation of the application, and do not constitute an improper limitation of the application.

[0023] Figure 1 Fig. 1 is a graph showing the changes of the body weight and liver coefficient of mice in the embodiment of the present application; 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 Fig. 2 is a graph showing the morphological changes of the liver of mice in the embodiment of the present application;

[0025] Figure 3 Fig. 3 is a graph showing the pathological structure changes of the liver of mice in the embodiment of the present application;

[0026] Figure 4 Fig. 4 is a graph showing the fatty degeneration of the liver of mice in the embodiment of the present application;

[0027] Figure 5 Fig. 5 is a graph showing the pathological structure changes of the liver of mice in the embodiment of the present application;

[0028] Figure 6 Fig. 6 is a graph showing the levels of Fasn, Cpt1a, Timp1 and Gck in the liver tissue of mice in the embodiment of the present application; 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 Fig. 7 is a graph showing the oil red staining results of AML12 cells in the embodiment of the present application;

[0030] Figure 8 Figure 6 is a graph showing the change of glucose content in the culture medium of AML12 cells in the embodiments of the present application; *P<0.5 compared with the control group; P<0.5 compared with the high-fat high-sugar group, # P<0.5.

[0031] Figure 9 Figure 8 is a graph showing the levels of Collagen 1, Fasn, Hmgcr and G6pc1 in AML12 cells in the embodiments of the present application; *P<0.5, **P<0.1, ***P<0.01 compared with the control group; P<0.5, P<0.1, P<0.01 compared with the high-fat high-sugar group, # P<0.5, ## P<0.1, ### P<0.01. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed description is illustrative only, and is intended to provide further description in order to provide a fuller enabling teaching of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0033] It is also important to note that the terms used herein are not intended to limit the particular embodiments of the present application to the preferred embodiments described. Rather, the terms are used only to describe specific embodiments of the present application. As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" are intended to include the plural forms as well, i.e., the inclusion of "a" or "an" or "the" means "one or more" unless the context clearly indicates otherwise. Furthermore, it is to be understood that the use of the term "including," "including," "includes," "has," "having," "comprising," "comprises" or "comprised of," "containing," "contains" or "contain," or variants or combinations thereof, in the detailed description and / or in the claims, is intended to denote the presence of stated features, steps, operations, devices, components, and / or groups thereof, but does not preclude the presence or addition of one or more other features, steps, operations, devices, components, and / or groups thereof.

[0034] In one exemplary embodiment of the present application, there is provided the use of pioglitazone in combination with ligustilide in any one or more of the following:

[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 sugar metabolism;

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

[0039] In the present application, the product can be a pharmaceutical or non-pharmaceutical test reagent, which can be used in basic research of liver fibrosis, particularly metabolic-related fatty liver fibrosis, such as constructing relevant animal or cell models, thereby being used in mechanism research of metabolic-related fatty liver fibrosis, etc.

[0040] In the present application, the liver fibrosis also includes complications mediated by liver fibrosis, further, the liver fibrosis is metabolic-related fatty liver fibrosis, and more specifically can be metabolic-related fatty liver fibrosis induced by high-fat and high-sugar, which is a pathological process of progressive liver injury accompanied by glucose and lipid metabolism disorder.

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

[0042] Specifically, at least the following effects are shown: reducing high-fat and high-sugar induced liver fat lesions; improving the pathological condition of liver tissue, so that the arrangement of liver cells tends to be normal, the number of fat vacuoles is reduced; the degree of fatty degeneration is reduced, the number of lipid droplets is reduced; regulating the glucose and lipid metabolism disorder process represented by key genes Fasn, Hmgcr, Cpt1a, Timp1, Gck, G6pc1, Collagen1; inhibiting the process of liver fibrosis induced by high-fat and high-sugar.

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

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

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

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

[0047] The non-pharmaceutically active ingredients can be carriers, excipients and diluents commonly used in pharmacy, etc. Moreover, according to the usual method, it can be made into powder, granules, suspension, emulsion, syrup, spray, etc. Oral agent, external agent, suppository and sterile injection solution form of dosage form are used.

[0048] The non-pharmaceutically active ingredients such as carriers, excipients and diluents that can be contained are well known in the art, and those skilled in the art can determine that they meet the clinical standards.

[0049] In still another embodiment of the present application, the carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil.

[0050] In still another embodiment of the present application, the pharmaceuticals of the present application can be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Such administration can be performed via single or multiple doses. It is understood by those skilled in the art that the actual dose to be administered in the present application 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 route of administration, the mode of administration, and the like.

[0051] In still another embodiment of the present application, the subject of the pharmaceutical administration can be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, and the like.

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

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

[0054] The "effective amount" refers to that amount of an active compound or pharmaceutical agent, including the compounds of the present application, which elicits the biological or medical response that is being sought in a tissue system, animal or human by a researcher, veterinarian, medical doctor or other medical person, including alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated. It will be appreciated that the optimal administration dosages and intervals of the active ingredients of the present application will depend on the nature and extent of the condition being treated, as well as the form, route and site of administration, and the particular mammal being treated, and this is within the skill of the art. The optimal administration dosage can be determined by routine experimentation.

[0055] The present application is further illustrated by the following specific examples, which are not intended to be limiting in any way. Any simple modification, equivalent change and modification made to the embodiments according to the technical essence of the present application are within the scope of the technical solutions of the present application.

[0056] Examples

[0057] I. Experimental method

[0058] 1. Construction of in vivo model

[0059] 1.1 Experimental animal modeling and drug administration

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

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

[0062] (2) Experimental animal modeling and drug administration: except for the control group, the rest of the experimental mice were given high-fat feed (42% Kcal fat and 0.2% cholesterol, TD88137), high-sugar water (23.1 g / L d-fructose + 18.9 g / L d-glucose), and free drinking for six weeks. After six weeks, each mouse was given drug administration by gavage at 1 ml / 100 g body weight, and the control group and high-fat high-sugar group were given the same volume of normal saline.

[0063] 1.2 Experimental animal sampling

[0064] The mouse body weight was measured every week, and the body weight value was recorded. After the mice were anesthetized, the blood was collected by inferior vena cava method, and when the blood volume was sufficient, the mice were sacrificed by cervical dislocation and abdominal dissection. The entire liver was cut off and placed in a culture dish, the liver morphology was photographed, and the liver weight value was recorded. The same part of the liver was taken and placed in the tissue fixative for subsequent morphological detection, and the remaining liver tissue was placed in the corresponding cryopreservation tube, quickly frozen with liquid nitrogen, and stored at -80°C for later use.

[0065] 1.3 Morphological detection of liver tissue

[0066] Mouse liver tissue hematoxylin-eosin (H&E) staining 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%), transparent agent: xylene, embedding agent: paraffin, staining agent: hematoxylin and eosin (0.5%-1% eosin solution), differentiation solution: 1% hydrochloric acid ethanol, blue solution: Scott's blue solution, mounting agent: neutral gum. Microtome, glass slide, cover glass, staining jar, oven, microscope.

[0069] 1.3.2 Experimental methods

[0070] (1) Tissue fixation: A cube-shaped liver tissue block with a side length of 5 mm was obtained from the liver lobe near the portal vein of the mouse liver tissue and immediately placed in 4% neutral paraformaldehyde for fixation for 24-48 h.

[0071] (2) Dehydration and clearing: After fixation, the liver tissue was dehydrated in 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 in xylene twice, each time for 1 h.

[0072] (3) Paraffin embedding: Immerse the transparent tissue in melted paraffin (65°C) twice, each time for 1 hour. Then, place the tissue in an embedding box and cool it to solidify.

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

[0074] (5) Dewaxing and hydration: The tissue sections were placed in xylene for dewaxing, 3 times, 5 minutes each time, and then

[0075] Gradient hydration was performed using 100% ethanol (5 min, three times) - 95% ethanol (5 min) - 80% ethanol (5 min) - 70% ethanol (5 min) - distilled water (5 min).

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

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

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

[0079] (9) Dehydration and clearing: The sections were dehydrated and cleared in a gradient manner, sequentially through 70% ethanol (30 seconds) - 80% ethanol (30 seconds) - 90% ethanol (30 seconds) - 95% ethanol (1 min) - 100% ethanol (1 min) - 100% ethanol (1 min) - xylene (2 min) - xylene (2 min).

[0080] (10) Covering and observation: 1-2 drops of neutral gum were added to the section, a cover glass was covered, and after air drying at room temperature, Aperio Versa super-resolution imaging instrument was used for mouse liver tissue H&E staining observation and image acquisition.

[0081] The NAFLDActivity Score (NAS) scoring system proposed by the American Nonalcoholic Steatohepatitis Clinical Research Network (NASH-CRN) in 2005 was used to evaluate the liver tissue, which evaluated the degree of fatty degeneration, lobular inflammation and ballooning of the liver tissue of each group of mice, and the higher the score, the higher the score. The standard is shown in Table 1.

[0082] Table 1 NAS scoring standard

[0083]

[0084]

[0085] 1.4 Liver tissue fat differentiation morphological detection

[0086] The expression of adipose differentiation-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 method

[0090] (1) De-waxing and hydration: as described in H&E staining, after the liver tissue section was baked in a 60°C oven until the paraffin melted, it was de-waxed with xylene (3 times, 5 min each time) and hydrated with gradient ethanol.

[0091] (2) Antigen repair: immerse the section in preheated antigen repair solution (sodium citrate buffer), heat in water bath to 85°C, keep for 40 min, after the end, cool to room temperature, rinse with distilled water for 5 min.

[0092] (3) Blocking: prepare a blocking solution containing 0.2% Triton X-100 in 2.5% BSA PBS, and prepare a 10% goat serum solution with the blocking solution, add to the section and block at room temperature for 30 min, which is used to block non-specific binding sites.

[0093] (4) Primary antibody incubation: Add dropwise the anti-ADRP antibody (1:200) diluted with blocking solution, place in a wet box, and incubate overnight in a refrigerator at 4°C. After completion, wash with 1XPBS for 3 times, each for 3 min.

[0094] (5) Secondary antibody incubation: Add dropwise the rabbit anti- Alexa Fluor 594 fluorescent labeled secondary antibody prepared with blocking solution onto the tissue sections, avoid light, and incubate at room temperature for 1 h. After completion of incubation, wash the sections with PBS for 3 times, each for 3 min.

[0095] (6) Mounting and image acquisition: Mount with DAPI containing quenching agent, and use laser scanning confocal microscope (40x) to acquire fluorescent images.

[0096] 1.5 Liver tissue fibrosis detection

[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 method

[0101] (1) De-waxing and hydration: As described in H&E staining, after the liver tissue sections are baked in an oven at 60°C until the paraffin melts, de-waxing with xylene (3 times, each for 5 min) and gradient ethanol (100% ethanol for 3 min, 95% ethanol for 3 min, 70% ethanol for 3 min) hydration, and finally in distilled water for 5 min.

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

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

[0104] (4) Dehydration and mounting: sequentially pass the sections through 70% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 3 min, xylene for 2 min, xylene for 2 min, and xylene for 2 min, and then mount with neutral resin, dry at room temperature, and then use Aperio Versa super-resolution imager to observe the staining and acquire images.

[0105] 1.6 Liver tissue glycolipid metabolism related gene expression

[0106] The mRNA levels of Fasn, Cpt1a, Timp1, and Gck, which are related to sugar and lipid metabolism, were detected in mouse liver tissues 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 (sterile and enzyme-free water preparation), sterile and enzyme-free water, Nanodrop spectrophotometer, real-time fluorescent quantitative PCR instrument.

[0109] 1.6.2 Experimental method

[0110] (1) RNA extraction: 100 mg of liver tissue was added to 700 μl of Trizol reagent, homogenized thoroughly, then 200 μl of chloroform was added, vortexed for 10 times, 2 s each time, until completely mixed, and then placed at room temperature for 10 min. 4°C, 12000g, centrifugation for 15 min, transfer the upper transparent layer to another new EP tube, add equal volume of isopropanol, upside down for 20 times, room temperature for 15 min. 4°C, 12000g, centrifugation for 10 min, discard the supernatant, visible RNA precipitate, add 900 μl 75% alcohol (sterile and enzyme-free water preparation), upside down once, 4°C, 7500g, centrifugation for 5 min, discard the supernatant, open the cover and lie in the EP tube to dry the reagent, add 45°C preheated sterile and enzyme-free water to dissolve the RNA, and use the ultramicro UV-visible spectrophotometer (NanoDrop One) to detect the RNA concentration.

[0111] (2) Reverse transcription: according to the reverse transcription kit instructions, the total amount of RNA is 1 μg, and the system is 20 μl. According to the RNA concentration, calculate the amount of RNA and the volume of sterile and enzyme-free water required. Sterile and enzyme-free water, RNA, 4 μl 4×gDNAwiper mix were added to 200 μl EP tube in turn, 42°C reaction for 2 min; then add 4 μl 5×HisScript III qRTSuper mix, 37°C reaction for 15 min, finally 5 seconds maintenance at 85°C.

[0112] (3) cDNA amplification and quantification: The cDNA stock solution obtained by reverse transcription was diluted to 2 ng / μl. The reaction system of corresponding primers was configured 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 sterile water + 0.4 μl of primers). Hprt1 was used as an internal reference for detection, and the amplification program was set in the real-time fluorescent quantitative PCR instrument: 95°C for 10 min, followed by 39 cycles of 95°C for 15 s and reduction to 60°C for 30 s. The copy number was read and recorded, and the relative fold change of the expression amount of the target gene mRNA was calculated: sample mRNA relative expression amount = Power [2, (sample target gene copy number - sample Hprt1 gene copy number) / (control group target gene copy number - control group Hprt1 gene copy number)].

[0113] 2. Construction of in vitro model

[0114] 2.1 Cell modeling and drug administration

[0115] AML12 (mouse normal liver cells) 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 inoculated in a six-well plate at a density of 500,000 per well. The experimental groups were: control group, high-fat high-sugar group, high-fat high-sugar + ligustilide group (20 μM), high-fat high-sugar + pioglitazone group (10 μM), high-fat 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 cultured in a cell incubator for 1 h. Then, high-fat high-sugar (palmitic acid + glucose, final concentration 300 μM + 10 mM) was added to the rest of the groups except the control group, and the corresponding concentration of drugs was added. After 24 h, samples were collected according to different experimental purposes.

[0116] 2.2 Oil red staining of AML12 cells

[0117] 2.2.1 Experimental materials

[0118] Isopropyl alcohol, oil red O staining solution (0.5 g of oil red O powder was dissolved in 100 ml of isopropyl alcohol to obtain an oil red O stock solution, then the oil red O stock solution was mixed with distilled water at a ratio of 3:2 to obtain an oil red O working solution, which was filtered after standing for 10 min and stored at 4°C in the dark), 4% paraformaldehyde fixing solution, inverted fluorescence microscope.

[0119] 2.2.2 Experimental method

[0120] (1) Cell fixation: After the end of cell treatment, discard the culture medium, gently wash the cells with PBS twice, add 4% paraformaldehyde fixing solution, and fix the cells at room temperature for 30 min. Then wash the cells with PBS twice, each for 3 min.

[0121] (2) Oil red O staining: After cell fixation, discard PBS, add oil red O working solution, and stain at room temperature for 30 min in the dark. Remove the staining solution and quickly rinse with 60% isopropanol (5-10 seconds) to remove excess staining solution. Then wash with PBS twice, each for 3 min. Observe the staining results and acquire images using an inverted fluorescence microscope.

[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, constant temperature oven.

[0125] 2.3.2 Experimental method

[0126] (1) Sample processing: Take the cell culture from the incubator and collect the supernatant. Centrifuge at 1500 rpm for 10 min to remove cells and debris, and take the supernatant for subsequent detection.

[0127] (2) Preparation of glucose oxidase reagent: Prepare the reagent 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), and peroxidase (8 kU / L) using distilled water as the solvent, and store at 4°C in the dark.

[0128] (3) Standard curve preparation and sample detection: Take 10 μL of distilled water (blank wells), 10 μL of standard (standard wells), and cell culture supernatant (sample wells) in a 96-well plate, add 250 μL of glucose oxidase reagent to each well, gently shake the plate, and incubate in a 37°C constant temperature incubator for 10 min. After incubation, measure the absorbance value of each well at a wavelength of 505 nm using a microplate reader, and record the value as A.

[0129] (4) Calculation of content: Glucose content (mmol / L) = [(A assay - A blank) / (A standard - A blank)] * 5.55.

[0130] 2.4 Expression of Glycolipid Metabolism-Related Genes in AML12 Cells

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

[0132] 2. Experimental Results

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

[0134] like Figure 1 As shown, the experimental results show that the weight of the mice in the control group increased steadily during the 16-week feeding period, the weight gain was within the normal range, and the growth curve was relatively gentle. The weight of the mice in the high-fat and high-sugar model group increased rapidly, indicating that the high-fat and high-sugar diet caused a significant increase in the weight of the mice. After treatment with ligustilide, pioglitazone, and the combination of the two drugs, the weight gain of the mice was significantly reduced. At the same time, compared with the control group, the liver coefficient of the mice in the high-fat and high-sugar model group was also significantly increased, while ligustilide and the combination of the two drugs reduced the liver coefficient of the mice, among which the reduction effect of pioglitazone and ligustilide combination was the most significant, indicating that the liver damage induced by high fat and high sugar was alleviated.

[0135] 2. Changes in Mouse Liver Morphology

[0136] like Figure 2 As shown, the experimental results showed that compared with the control group, the livers of mice in the high-fat and high-sugar group showed obvious pathological characteristics, with increased liver volume, grayish-white color, blunt edges, and a fatty luster on the surface, indicating that the liver showed obvious fatty degeneration due to excessive lipid accumulation. In addition, slight unevenness or punctate congestion can be seen, which is related to tissue structure destruction caused by lipotoxic damage and inflammatory response. After treatment with ligustilide, pioglitazone, and the combination of the two drugs, the morphology of the mouse liver was significantly improved, and 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 boundaries were clear, indicating that these drugs can effectively reduce liver fatty lesions induced by high fat and high sugar, and the improvement effect of the combination of pioglitazone and ligustilide was the most significant, and the liver morphology was close to that of the control group.

[0137] 3. Pathological structural changes in mouse liver

[0138] like Figure 3As shown in Table 2, the NAS score of the H&E liver of mice in the high-fat, high-glucose group was 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 reduced the NAS score. The combination of ligustilide and pioglitazone reduced the NAS score to a greater extent than either the single drug or the sum of the two drugs, indicating a synergistic effect.

[0139] Table 2 NAS score of liver of mice in each group

[0140]

[0141] 4. Hepatic steatosis in mice

[0142] like Figure 4 As shown in the results, compared with the control group, the accumulation of lipid droplets in the liver tissue of mice in the high-fat and high-sugar group increased significantly, which was manifested as a strong green fluorescence signal. The positive staining area reached 34.01±3.81%, and the distribution was clustered, indicating that the high-fat and high-sugar treatment led to a significant increase in fatty degeneration of the mouse liver. After treatment with ligustilide (21.56±2.12%), pioglitazone (23.05±1.86%) and the combination of the two (9.88±1.95%), the degree of fatty degeneration of the mouse liver was effectively reduced, the positive fluorescence staining area was significantly reduced, and the number of lipid droplets was reduced. As shown in Table 3, compared with the high-sugar and high-fat group, the fluorescence intensity of the ligustilide monotherapy group decreased by 36.61%, the fluorescence intensity of the pioglitazone monotherapy group decreased by 32.20%, and the combination of the two decreased by 70.95%, which was more significant and restored to a level close to normal.

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

[0144]

[0145]

[0146] 5. Pathological structural changes in mouse liver

[0147] like Figure 5As shown in the experimental results, in the high-fat, 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 sinusoids and portal areas, indicating that the high-fat, high-sugar diet induced a significant increase in extracellular matrix and the occurrence of fibrosis in the liver. After treatment with ligustilide, pioglitazone, and the combination of the two drugs, the deposition of collagen fibers in the liver of mice was reduced, and the effect of the combination of pioglitazone and ligustilide was more significant, and the liver tissue structure was closer to that of the normal group. This shows 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 combination of drugs has a stronger anti-fibrotic effect. The specific staining positive area statistics are shown in Table 4. Compared with the high-fat, high-sugar group, the collagen fiber deposition area in the ligustilide monotherapy group decreased by 43.18%, the positive area in the pioglitazone monotherapy group decreased by 32.54%, and the combined use of the two drugs decreased by 79.47%, which is more significant and restored to near normal levels.

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

[0149]

[0150] 6. Changes in Fasn, Cpt1a, Gck, and Timp1 Levels in Mouse Liver Tissue

[0151] like Figure 6 As shown, the experimental results showed that compared with the control group, the expression of the fatty acid synthase-related gene Fasn, the glucose metabolism regulatory gene Gck, and the collagen degradation-related gene Timp1 in the liver tissue of mice in the high-fat, high-glucose group was significantly upregulated, while the expression of the carnitine palmitoyltransferase gene Cpt1a was significantly downregulated. This indicates that high-fat, high-glucose stimulation leads to liver glucose and lipid metabolism disorders and extracellular matrix deposition in mice. Ligustilide, pioglitazone, and their combination can all downregulate the expression levels of Fasn, Gck, and Timp1, and upregulate the expression of Cpt1a, with the regulatory effect of pioglitazone and ligustilide combination being more significant. This indicates that both ligustilide and pioglitazone can improve liver glucose and lipid metabolism disorders induced by high-fat, high-glucose diet by inhibiting lipid synthesis, promoting lipid metabolism, and regulating glucose metabolism, and that the combination of pioglitazone and ligustilide has a more synergistic effect.

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

[0153] like 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 accumulate in the cytoplasm in the high-fat and high-sugar model group, indicating that the intracellular lipid deposition is significantly increased, reflecting that high fat and high sugar induce lipid metabolism disorders in AML12 cells. After treatment with ligustilide, pioglitazone and the combination of the two drugs, the number and volume of red lipid droplets in the cells were significantly reduced, and lipid deposition was significantly alleviated, among which the effect of the combination of pioglitazone and ligustilide was more prominent. The specific statistics of the positive staining area are shown in Table 5. Compared with the high sugar and high fat group, the collagen fiber deposition area of ​​the ligustilide monotherapy group decreased by 33.17%, the positive area of ​​the pioglitazone monotherapy group decreased by 36.26%, and the combination of the two decreased by 79.82%, which was more significant and restored to a level close to normal.

[0154] Table 5 Statistics of Oil Red Staining Positive Areas of AML12 Cells in Each Group

[0155]

[0156] 8. Changes in Glucose Content in AML12 Cell Culture Medium

[0157] like Figure 8 As shown in the experimental results, compared with the control group, the glucose content in the AML12 cell culture medium in the high-fat and high-sugar group was significantly increased, indicating that AML12 cells had a glucose metabolism disorder. The high-sugar and high-fat environment led to impaired glucose uptake in AML12 cells and induced insulin resistance. Treatment with ligustilide, pioglitazone, and their combination all reduced the glucose content in the culture medium and effectively improved abnormal cellular glucose metabolism. The combination of pioglitazone and ligustilide was more effective.

[0158] 9. Changes in Collagen1, Fasn, Hmgcr, and G6pc1 Levels in AML12 Cells

[0159] like Figure 9 As shown in the experimental results, compared with the control group, the high-fat, high-glucose model significantly upregulated the expression of Fasn, the cholesterol synthesis-related gene Hmgcr, and the glucose metabolism gene G6pc1 in AML12 cells. This also upregulated the expression of the collagen-related gene Collagen1. This suggests that high-fat, high-glucose stimulation leads to disrupted glucose and lipid metabolism in AML12 cells, triggering an increase in extracellular matrix. Ligustilide, pioglitazone, and their combination can all downregulate the expression of these key glucose and lipid metabolism genes, with the pioglitazone-ligustilide combination having a more pronounced downregulation effect.

[0160] To sum up, the application carries out comprehensive research on the drug composition through the constructed in-vivo and in-vitro fatty liver fibrosis evaluation system, and confirms that the drug composition exhibits more significant treatment effect compared with the existing single drug therapy, thereby providing a new solution for the prevention and treatment field of metabolic related fatty liver fibrosis.

[0161] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Various modifications and changes can be made by those skilled in the art based on the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of pioglitazone combined with ligustilide in the preparation of a medicament for preventing and / or treating liver fibrosis; The mass ratio of pioglitazone to ligustilide is 1:1; The liver fibrosis is metabolism-related fatty liver fibrosis mediated by high fat and high sugar.

2. A pharmaceutical composition for preventing and / or treating liver fibrosis, characterized in that: The active ingredients of the pharmaceutical composition at least include pioglitazone and ligustilide; the mass ratio of pioglitazone to ligustilide is 1:1; The liver fibrosis is metabolism-related fatty liver fibrosis mediated by high fat and high sugar.

3. The pharmaceutical composition according to claim 2, wherein The pharmaceutical composition further comprises at least one non-pharmaceutically active ingredient.

4. The pharmaceutical composition according to claim 3, wherein The non-drug active ingredient is a carrier commonly used in pharmacy.

5. The pharmaceutical composition according to claim 2, wherein The subjects of drug administration are humans and non-human mammals.

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