CN117982486A

CN117982486A - Application of wogonin derivative V8 in preparation of medicines for preventing and treating liver fat metabolic disorder related diseases

Info

Publication number: CN117982486A
Application number: CN202410092226.2A
Authority: CN
Inventors: 张小博; 魏立彬; 周煜新; 王卓; 韩晓蕾; 高源�
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2024-01-23
Filing date: 2024-01-23
Publication date: 2024-05-07

Abstract

The invention discloses application of wogonin derivative V8 in preparation of medicines for preventing and treating liver fat metabolism disorder related diseases, wherein the liver fat metabolism disorder related diseases are fatty liver, liver fibrosis, liver cirrhosis and liver cancer. According to the invention, through constructing a cell fatty liver model, a mouse liver fibrosis model and a tumor model driven by mouse primary chronic inflammation, the wogonin derivative V8 treatment is proved to be capable of remarkably reducing abnormal accumulation of cell fat, reducing damage and fibrosis degree of mouse liver cells, promoting lipid metabolism of liver tissues and inhibiting release of inflammatory factors, remarkably reducing tumor incidence, and not damaging blood system cells after continuous administration. This shows that wogonin derivative V8 can improve the relevant pathological characteristics caused by liver lipid metabolism disorder and is used for preparing medicines for improving liver fat metabolism disorder related diseases.

Description

Application of wogonin derivative V8 in preparation of medicines for preventing and treating liver fat metabolic disorder related diseases

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of wogonin derivative V8 in preparation of a medicine for preventing and treating liver fat metabolic disorder related diseases.

Background

The liver is an important organ of body fat metabolism, and its normal functioning is important for maintaining body fat metabolic homeostasis. The bad life style, disease or environmental factors can lead to liver fat metabolism disorder, cause abnormal accumulation of liver cell lipid, generate cytotoxicity and tissue inflammation, and represent clinically common chronic diseases such as fatty liver disease, liver fibrosis, liver cirrhosis, liver cancer and the like according to different pathological progress. With the global rising incidence of various metabolic syndromes (e.g., obesity, diabetes, hypertension, hyperlipidemia, etc.), body metabolic disorders also further affect liver fat metabolism, e.g., 80-90% of obese patients and 70-80% of type 2 diabetics are accompanied by liver lipid metabolism abnormalities. Therefore, the development of the medicine for improving liver fat metabolism not only has important significance for fatty liver diseases, but also can effectively reduce the incidence rate of fatty liver related to metabolic diseases.

The compound V8 is a newly synthesized derivative of wogonin which is an effective active ingredient of the traditional Chinese medicine baicalein, the structural formula of the derivative is shown in the following formula, and in terms of structure, V8 introduces a tertiary amine side chain with hydroxyl into a flavone mother ring of the original wogonin, so that the derivative has better water solubility.

Only a few studies in the early stage find that the compound can have a certain antitumor potential, but no study on other diseases is reported. Therefore, the application of V8 in preparing medicaments for preventing and/or treating liver metabolic diseases such as fatty liver, liver fibrosis, liver cirrhosis, liver cancer and the like is not seen at present.

Disclosure of Invention

The invention aims to provide application of wogonin derivative V8 in preparation of medicines for preventing and treating liver fat metabolic disorder related diseases.

Further, the liver fat metabolism disorder related diseases are fatty liver, liver fibrosis, liver cirrhosis and liver cancer.

Further, the anti-drug is a pharmaceutical composition composed of an active ingredient wogonin derivative V8 and pharmaceutically acceptable auxiliary materials.

Further, the dosage form of the pharmaceutical composition is tablets, granules, pills, capsules or injections.

According to the invention, through constructing a cell fatty liver model, a mouse liver fibrosis model and a tumor model driven by mouse primary chronic inflammation, the wogonin derivative V8 treatment is proved to be capable of remarkably reducing abnormal accumulation of cell fat, reducing damage and fibrosis degree of mouse liver cells, promoting lipid metabolism of liver tissues and inhibiting release of inflammatory factors, remarkably reducing tumor incidence, and not damaging blood system cells after continuous administration. This shows that wogonin derivative V8 can improve the relevant pathological characteristics caused by liver lipid metabolism disorder, so wogonin derivative V8 is proposed to be used for preparing medicines for improving liver lipid metabolism disorder related diseases.

Drawings

FIG. 1 shows the effect of V8 on cell viability.

FIG. 2 shows the effect of V8 on intracellular lipid accumulation.

FIG. 3 shows the effect of V8 on liver fibrosis in mice.

FIG. 4 shows the effect of V8 on pre-cancerous pathology in the liver of mice.

FIG. 5 shows the effect of V8 on inflammatory factor release and liver lipid deposition.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1

1. Experimental materials

1.1 Test drug

Name: v8

The content is as follows: >98%

Providing units: university of Chinese medicine science

The preparation method comprises the following steps: for cell experiments, preparing the drug powder into a mother solution with the concentration of 30mM by using dimethyl sulfoxide (DMSO), and storing at the temperature of-80 ℃ for later use; for animal experiments, the drug was formulated as a 40mg/mL suspension using a 0.5% sodium carboxymethylcellulose (CMC-Na) solution and placed at 4℃for use.

1.2 Cell culture

Both the human liver cancer cell line HepG2 and the human normal liver cell line L02 were purchased from Shanghai cell bank of China academy of sciences, and cultured in a DMEM medium containing 100U/mL penicillin, 100mg/mL streptomycin and 10% fetal bovine serum in a constant temperature incubator at 37℃with 5% CO ₂.

1.3 Laboratory animals

Source, germ line, strain: ICR mice were purchased from Hangzhou medical college (laboratory animal production license: SCXK (Zhejiang) 2019-0002).

Week-old: 2 weeks of age and 6-8 weeks of age

Gender: and (5) male.

2. Cell experiment

2.1 Free Fatty Acid (FFA) induced cellular fat accumulation model

DMEM cell culture broth containing 1% Bovine Serum Albumin (BSA) was first prepared, and then a mixture of Oleic Acid (OA) and Palmitic Acid (PA) (OA: pa=2:1) was added to a final concentration of 0.5mM. Cells were cultured using this solution for 12 hours, thereby inducing cell fat deposition.

2.2 Apoptosis detection

Cells in the logarithmic growth phase were inoculated at a density of 2X 10 ⁵ cells/well into six-well plates, and after overnight incubation, control and FFA treatments were added for 12h, respectively, followed by further treatment with different concentrations of V8 compound for 24h. After cells were collected by digestion with pancreatin without EDTA, the cells were washed twice with PBS, 100. Mu.L of 1 Xbinding Buffer was added to each sample, 5. Mu.L of each of Annexin V-FITC and PI staining solution was added, gently mixed, and incubated at room temperature for 10min in the absence of light. Finally, 400. Mu.L of 1 Xbinding Buffer was added, and after mixing, the mixture was examined by a flow cytometer.

2.3 Cell oil Red O staining

Cells were fixed with 4% paraformaldehyde solution for 10 minutes, then washed twice with PBS, added with an appropriate amount of oil red O staining solution for 30 minutes, washed with washing solution after the staining solution was discarded, washed with PBS, finally added with an appropriate amount of PBS, and the lipid droplet size was observed under a microscope. After the observation, the stained cells were lysed with isopropanol, and the lipid deposition was quantified by adding the isopropanol solution after the lysis of the lipid droplets to a 96-well plate, and detecting the absorbance at 520nm wavelength.

2.4 Statistical analysis

The experimental results were expressed in mean±sd and significance was detected by one-way analysis of variance using GRAPHPAD PRISM software. Ns represents no difference compared to the blank group, P <0.05, P <0.01, P <0.001; ns represents no difference, # represents P <0.05, # represents P <0.01, and # # represents P <0.001, compared to the negative control group.

2.5 Experimental results

V8 reduces cellular fat accumulation

In the cell model of FFA-induced cellular lipid accumulation, V8 cytotoxicity was first examined using two cell lines (L02 and HepG 2). As shown in fig. 1, the V8 compound concentration did not significantly differ between the cell viability of the treated cells within 30 μm, and thus did not cause a cytotoxic effect. Oil red O staining, in turn, showed a significant increase in lipid accumulation in cells following FFA addition, whereas V8 treatment significantly reduced lipid levels in cells and was concentration dependent. This result was verified in both the human liver normal cell line L02 and the liver cancer cell line HepG2 (shown in FIG. 2). Furthermore, V8 itself in the experiment had no significant effect on cell viability, indicating that this effect was not due to increased cell death.

3. Animal experiment

3.1CCl ₄ Induction of mouse liver fibrosis model

ICR mice (body weight: 18-20 g) of 6-8 weeks old without specific pathogen were intraperitoneally injected with 10% CCl ₄ (olive oil diluted) solution (0.5 mL/kg) twice weekly for 8 weeks. Drug treatment was started at week 5 of cci ₄ injection and continued for 4 weeks until the end of the experiment.

The experimental animals were grouped and the dose set as follows:

Group of	Medicament	Dosage of	Administration mode	Number of animals
					Blank control group	/	/	/	3
Negative control group	Physiological saline	/	Once every two days p.o	8
					Drug treatment group	V8	100mg/kg	Once every two days p.o	8
Drug treatment group	V8	200mg/kg	Once every two days p.o	8

3.2DEN/CCl ₄ Induction of Primary liver cancer in mice model

Two week old ICR mice were intraperitoneally injected with DEN (25 mg/kg) once, and four weeks later, the injection was continued once. Mice were intraperitoneally injected with 10% cci ₄ (olive oil diluted) solution (0.5 mL/kg) once a week for 22 weeks from the third week of age. Drug treatment was started after the end of injection cci ₄ for 8 weeks to the end of the experiment.

The experimental animals were grouped and the dose set as follows:

Group of	Medicament	Dosage of	Administration mode	Number of animals
					Negative control group	Physiological saline	/	Once every two days p.o	5
Drug treatment group	V8	200mg/kg	Once every two days p.o	5
					Drug treatment group	V8	400mg/kg	Once every two days p.o	5

3.3 Blood cell detection

After the administration, the orbit of the mouse is sampled, placed in an anticoagulation tube and detected by a hemocytometer.

3.4 Staining and scoring of tissue sections

After the fresh liver tissue of the mice is fixed by 4% paraformaldehyde, the fresh liver tissue is embedded into paraffin for slicing through dehydration-permeabilization treatment. The prepared paraffin sections were dewaxed and used for hematoxylin-eosin (HE) staining and Masson trichromatic staining, respectively. Liver pathology was scored using the METAVIR scoring system and liver collagen fiber accumulation was quantified using ImageJ software.

3.5 Serum ELISA detection

50. Mu.L of each of a standard (cytokine standard diluted with a buffer) and a sample to be tested (mouse serum diluted with a buffer) was added to the reaction plate, and 100. Mu.L of a detection antibody labeled with horseradish peroxidase (HRP) was added. The reaction plates were washed 5 times, 1min each at 37℃incubation 1h,washing buffer. Substrates a and B were mixed according to 1:1 volume was mixed well, 100. Mu.L of substrate mixture was added to each well, and incubated at 37℃for 15min in the absence of light. 50 μl of stop solution was added to each well, and the absorbance of each well was read on an microplate reader. And establishing a standard curve according to the concentration and absorbance of the standard substance, so as to calculate the actual concentration of the corresponding cytokine in the sample to be detected.

3.6 Liver Triglycerides detection

Accurately weighing a tissue sample, wherein a homogenizing medium is normal saline, and the weight (g): homogenization medium (mL) =1: 9, adding normal saline in proportion, homogenizing on ice, transferring to an EP tube after the homogenization, centrifuging at 2500rpm and 4 ℃ for 5min, and taking supernatant to be measured. Blank holes, standard holes and sample holes are arranged, and compound holes are arranged. 250 mu L of working solution is added into a 96-well plate, then 2.5 mu L of physiological saline, calibrator (triglyceride standard solution) and sample to be tested (tissue lysate) are respectively added, the mixture is uniformly mixed, the mixture is incubated for 10min at 37 ℃, and the absorbance value of each well is measured by an enzyme-labeled instrument at the wavelength of 510 nm. At the same time, each sample BCA was assayed for protein concentration, and the triglyceride content was calculated from the sample protein concentration (cprot) according to the following formula:

TG content (mmol/g prot) = (a _{Sample hole}-A_{Blank hole})/(A_{standard hole}-A_{Blank hole})×C_{Standard of} C prot.

3.7 Statistical analysis

3.8 Experimental results

(1) V8 inhibited CCl ₄ -induced liver fibrosis in mice

In the model of CCl ₄ -induced liver fibrosis in mice, CCl ₄ induced a significant increase in pathological changes such as inflammatory cell infiltration (HE staining) and collagen fiber deposition (Masson staining) of tissues compared to the normal group, whereas V8 treatment was reduced in response to pathological changes, as shown in fig. 3 a. Using Metavir scores, V8 was seen to significantly improve liver fibrosis (B in fig. 3), and quantification of Masson staining also indicated that V8 significantly inhibited liver fibrosis (C in fig. 3).

(2) V8 ameliorates liver precancerous pathological changes in mice

In the DEN/CCl ₄ -induced mouse primary liver cancer model, as shown in fig. 4, the pathological characteristics of the liver tumor of the model control group are obvious, a plurality of anisotropic hyperplasia nodules are generally observed on the surface of the liver, cytopathy is observed by histological staining, such as cytoplasms cavitation, increase of cytoplasms proportion and the like, and the Masson trichromatism staining shows that certain fibrosis occurs in tissues. V8 treatment significantly improved the characteristics of chemically induced pre-cancerous lesions of the liver, especially in high dose V8 treated mice with normal liver histologic morphology, with significantly reduced collagen fiber deposition (fig. 4A). In agreement with this, V8 treatment also significantly inhibited the number of liver-proliferation nodules, thus blocking the formation of primary liver cancer (fig. 4B).

(3) V8 reduces inflammatory factor release and liver lipid deposition

In the development of liver cancer, which is often accompanied by inflammatory reactions and liver lipid metabolism disorders, as shown in FIG. 5, V8 treatment down-regulates the levels of inflammatory factors such as IL-1β, IL-6, TNF- α in serum while significantly inhibiting the accumulation of triglycerides in liver tissue in DEN/CCl ₄ -induced mice primary liver cancer model.

(4) Effects of V8 on the blood System

In order to determine whether the high dose V8 administration was toxic to the mouse blood system, the mouse blood cell-related index was further analyzed, and as shown in the following table, the V8 treatment showed little difference in blood cell-related index from the physiological saline group, and even increased numbers of white blood cells and platelets.

The above results show that V8 has better safety, so the clinical open potential is large.

Claims

1. Application of wogonin derivative V8 in preparing medicine for preventing and treating liver fat metabolism disorder related diseases is provided.

2. The use according to claim 1, wherein the liver fat metabolism disorder-related disease is fatty liver, liver fibrosis, cirrhosis, liver cancer.

3. The use according to claim 2, wherein the anti-drug is a pharmaceutical composition consisting of wogonin derivative V8 as active ingredient and pharmaceutically acceptable excipients.

4. The use according to claim 3, wherein the pharmaceutical composition is in the form of a tablet, granule, pill, capsule or injection.