CN113559127A - Application of exendin denstrom aqueous extract in preparing medicine for treating hepatic fibrosis - Google Patents
Application of exendin denstrom aqueous extract in preparing medicine for treating hepatic fibrosis Download PDFInfo
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
- A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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Abstract
The invention provides an application of an exendin denstrom aqueous extract in preparing a medicament for treating hepatic fibrosis, belonging to the technical field of biological medicines. The exendin pekinensis can obviously improve the expression level of MMP-13 in the liver tissue of an animal and obviously reduce the expression level of TIMP-1, thereby promoting the degradation of ECM and reducing the degree of HF. Animal experiments show that the levels of HA, LN, PC-III, C-IV and TGF-beta 1 in the serum of a hepatic fibrosis model mouse are obviously reduced after the treatment of the exendin densa; the damage of the liver tissue structure is gradually improved, the degree of liver cell edema, fatty lesion and inflammatory cell infiltration is obviously reduced, the fibrous deposition, the vascular region or the central venous fibrous interval are obviously thinned compared with a model group, and the proliferation of connective tissue is obviously reduced.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of an exendin denstrom aqueous extract in preparing a medicine for treating hepatic fibrosis.
Background
Hepatic Fibrosis (HF) is a pathological change present in most chronic liver disease processes, mainly manifested by the hyperproliferation and deposition of extracellular matrix (ECM) within liver tissue, resulting in abnormal structural changes of liver tissue and affecting its normalPhysiological function[1]. The synthesis and degradation of ECM are closely related to matrix metalloproteinase-13 (MMP-13) and matrix metalloproteinase inhibitor-1 (TIMP-1), MMP-13 promotes ECM degradation, TIMP-1 inhibits ECM degradation by inhibiting the activity of MMP-13, thereby promoting the formation of HF[2]. At present, a medicine which can effectively improve the expression level of MMP-13 in liver tissues and obviously reduce the expression level of TIMP-1 in the liver tissues for treating HF is lacked.
Disclosure of Invention
The invention aims to provide application of an aqueous extract of exendin firmus in preparation of a medicament for treating hepatic fibrosis, wherein the exendin firmus can obviously improve the expression level of MMP-13 in animal liver tissues and obviously reduce the expression level of TIMP-1, thereby promoting ECM degradation and reducing the degree of HF.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of an exendin denstrom aqueous extract in preparing a medicament for treating hepatic fibrosis.
The invention also provides application of the exendin nodosum in preparing a medicament for improving MMP-13 expression level in animal liver and simultaneously reducing TIMP-1 expression level in animal liver.
Preferably, the dosage form of the medicament comprises an oral preparation.
Preferably, the oral preparation comprises a decoction.
Preferably, the preparation method of the decoction comprises the following steps:
mixing the exendin brueckii with water, and decocting to obtain the medicine for treating hepatic fibrosis.
Preferably, the mass ratio of the exendin mandshurica to the water is 4-5: 100.
preferably, after the decoction, the decoction is further concentrated, wherein the concentration ratio is (112.5-450): 1000.
preferably, the decocting temperature is 90-100 ℃; the decoction time is 30-40 min.
The invention provides application of an exendin denstrom aqueous extract in preparing a medicament for treating hepatic fibrosis, and the exendin denstrom aqueous extract can obviously improve the expression quantity of MMP-13 in animal liver tissues and obviously reduce the expression quantity of TIMP-1, thereby promoting ECM degradation and lightening the degree of HF. Animal experiments show that the levels of HA, LN, PC-III, C-IV and TGF-beta 1 in the serum of a hepatic fibrosis model mouse are obviously reduced after the administration of the drug (an aqueous extract of the Eremias densa) by intragastric administration; the damage of the liver tissue structure is gradually improved, the degrees of liver cell edema, fatty lesion and inflammatory cell infiltration are obviously reduced, fibrous deposition, the interval of the vascular tract or the central venous fiber are obviously thinned compared with a model group, and the proliferation of connective tissue is obviously reduced; MMP-13 expression is obviously increased in mouse liver tissue (P is less than 0.01), and TIMP-1 expression is obviously reduced (P is less than 0.01).
Drawings
FIG. 1 shows histopathological changes (HE X200) of liver tissues of mice in each group under a light microscope; wherein A is a blank group; b is a model group; c is an Anluo chemical fiber group; d is the low dose group of exendin pekoe; e is the medium dose group of exendin pekoe; f is the high dose group of exendin pekoe.
Detailed Description
The invention provides an application of an exendin denstrom aqueous extract in preparing a medicament for treating hepatic fibrosis.
The invention also provides application of the exendin nodosum in preparation of a medicine for improving MMP-13 expression level in animal liver.
The invention also provides application of the exendin nodosum in preparing a medicament for reducing the TIMP-1 expression level in animal livers.
In the invention, the exendin nodosum is commercially available from general, and in the specific implementation process of the invention, the exendin nodosum is purchased from an outpatient clinic of a traditional Chinese medicine hospital affiliated to Ningxia medical university.
In the present invention, the dosage form of the drug preferably includes an oral preparation; the oral preparation preferably comprises a decoction.
In the present invention, the preparation method of the decoction preferably includes the following steps:
mixing the exendin brueckii with water, and decocting to obtain the medicine for treating hepatic fibrosis.
In the invention, the mass ratio of the exendin zerumbet to water is preferably 4-5: 100.
in the invention, after the decoction, the decoction is preferably concentrated, and the concentration ratio is preferably (112.5-450): 1000, more preferably 225: 1000.
in the invention, the preferable temperature of the decoction is 90-100 ℃; the preferable time for decoction is 30-40 min.
In the specific implementation process of the invention, 45g of exendin firmus and 1000ml of distilled water are taken, decocted to boil and concentrated to 450ml, dregs are filtered out, 100ml of the dregs is taken out to obtain low concentration (0.1g/ml), the rest is continuously concentrated to 225ml, 100ml of the dregs is taken out to obtain medium concentration (0.2g/ml), and the rest is concentrated to 112.5ml of the dregs to obtain high concentration (0.4 g/ml).
In the present invention, the therapeutic method of the drug for treating hepatic fibrosis is oral administration.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1 Material
1.1 animals SPF grade male ICR mice 60, 4 weeks old, weight (20 + -2) g; provided by the laboratory animal center of Ningxia medical university, license number: SCXK (Nine) 2020-; all mice were housed in SPF-level animal houses, fed with water ad libitum, and subjected to the test after adaptive housing for 7 days.
1.2 the medicine and reagent are single-ingredient exendin media, purchased from outpatient service of traditional Chinese medicine hospital affiliated to department of Ningxia medical university, 45g of exendin media and 1000ml of distilled water are taken, decocted to boil and concentrated to 450ml, dregs of decoction are filtered out, 100ml of the dregs of decoction is taken out, namely the low concentration (0.1g/ml), the rest is continuously concentrated to 225ml, 100ml of the dregs of decoction is taken out, namely the medium concentration (0.2g/ml), and the rest is concentrated to 112.5ml, namely the high concentration (0.4 g/ml). Decocting into Chinese medicinal decoction with concentration of 0.1g/ml, 0.2g/ml, 0.4g/ml before use, and storing in refrigerator at-4 deg.C; carbon tetrachloride (specification: 500ml, xu zhou tianhong chemical limited); corn oil (specification: 500ml, Beijing Soilebao Tech Co., Ltd.); anluohuaxianwan pill (specification: 6 g/bag, Senlong pharmaceutical Co., Ltd.) is prepared by grinding into powder, preparing into 0.16g/ml solution with distilled water, and storing in refrigerator at-4 deg.C for use.
Mouse serum Hyaluronic Acid (HA), Laminin (LN), type III procollagen (PC-III), type IV collagen (C-IV) and transforming growth factor-beta 1 (TGF-beta 1) enzyme linked immunoassay kit (Shanghai reputation Biotechnology Co., Ltd.); rabbit anti-TIMP-1, rabbit anti-MMP 13 (Beijing Olympic Biotech Co., Ltd.).
1.3 instrument embedding machine (Hubei Tai Wei science and technology industries, Ltd.); slicer (laika microscopy systems limited); microplate reader (finland Labsystems Multiskan MS); plate washers (Thermo Labsystems, finland); a micro high-speed centrifuge (made in China); chemiluminescence analyzer (shanghai sky energy corporation).
2 method
2.1 grouping and modeling 60 4-week-old ICR mice were acclimatized for 7 days and randomized into a blank group (10) and a model group (50). Reference to molding methods[3]The model group mice are injected with 10% carbon tetrachloride solution (prepared according to the volume ratio of carbon tetrachloride to corn oil being 1: 9) in the abdominal cavity, the dose of each injection is 5ml/kg, twice a week and lasting for 8 weeks, and the hepatic fibrosis model is manufactured. The mice in the blank group were injected intraperitoneally with an equal volume of 0.9% saline twice a week for 8 weeks. After 8 weeks, 3 mice were randomly selected from each group and sacrificed, and four liver fibrosis and liver tissue HE staining tests were performed on the mice, which showed that CCL4 successfully establishes a mouse liver fibrosis model. Mice successfully modelled were randomly divided into model groups, silibinin groups and low, medium and high dose groups of exendin michigan.
2.2 the administration dose is equivalent by converting the daily dose of an adult into the surface area of a mouse[4]Single-ingredient exendin nodosum low dose of 1 g/kg-1·d-1Medium dose of 2 g/kg-1·d-1High dose 4 g/kg-1·d-1Anluo chemical fiber 100 mg/kg-1·d-1Each administration group was administered dailyThe administration was carried out once per gavage, 5ml/kg each time, and continuously for four weeks. The blank and model groups were given an equal volume of 0.9% saline once daily for four consecutive weeks.
2.3 general conditions of animals the mice of each group were observed and recorded daily for weight, fur, spirit, activity, diet, etc. during the experiment.
2.4 after the last gastric lavage of the liver histomorphosis of the mouse for 24 hours, the liver part of the mouse is fixed by 4 percent paraformaldehyde for 24 hours, and after the ethanol dehydration, the liver part is soaked in wax, embedded, sliced, unfolded, fished and baked. HE staining was performed to observe histopathological changes in the liver.
2.5 immunohistochemical method for detecting MMP-13 and TIMP-1 content in liver tissue sections used in the experiment were placed in a refrigerator at 4 ℃ overnight, dehydrated, heat-repaired, sealed, primary antibody incubated, secondary antibody incubated, DAB developed, hematoxylin counterstained, mounted, and observed under a microscope for photographing. Semi-quantitative analysis of MMP-13 and TIMP-1 positive results was performed under a 200-fold microscope using Image J software. Each specimen was observed at random for 6 fields, and the relative content of the antigen was expressed as the area of positive product/field area, and averaged.
2.6 detection of liver fibrosis in mouse serum by enzyme-linked immunosorbent assay and TGF-beta1And horizontally taking 1-2 ml of mouse apex cordis blood, standing, and centrifuging to prepare mouse serum. And (3) detecting the contents of HA, LN, PC-III, C-IV and TGF-beta 1 in the serum of the mouse by using a microplate reader.
2.7 statistical methods data processing SPSS 22.0 software was used, results were obtained
And (4) showing. Performing normal distribution test on the data, wherein the data are in accordance with normal distribution, single-factor analysis of variance (One-way ANOVA) is adopted for homogeneous variance data comparison, and SNK-q method test is adopted for multiple comparison among the mean values of a plurality of samples; non-normal distribution-nonconforming and variance-nonuniform data are subjected to non-parametric test of rank conversion by P<A difference of 0.05 is statistically significant.
3 results
3.1 in general condition experiments, the blank group of mice is found to have good growth state, acute response to external stimulation, vitality in eyes, bright hair, normal increase of diet and weight and no death condition; compared with the blank mice, the modeling mice grow slowly, move slowly, have no spirit in eyes, have yellow fur, and have hair-explosion phenomenon, inappetence and lighter weight in most mice in the later period of modeling than the blank mice in the same period; after 4 weeks of drug intervention, the body weight of mice in each group except the model group gradually recovered and increased, and the food intake and water intake were also increased to some extent.
3.2 serum hepatic fibrosis of each group of mice and TGF-beta1Horizontal change of state
The main pathological manifestations of hepatic fibrosis are the growth of hepatic collagen fibers, the deposition of liver extracellular matrix (ECM), HA, LN, PC-III and IV-C of the four hepatic fibers are the main components of the ECM, the levels of the HA, LN, PC-III and IV-C are closely related to the process of ECM accumulation and collagen crosslinking into fibers, and the accumulation degree of the ECM is indirectly reflected, so that the hepatic fibrosis degree can be judged by detecting the contents of HA, LN, PC-III and C-IV in serum; transforming growth factor (TGF- β) and receptors are secreted by Hepatic Stellate Cells (HSC), and TGF- β 1 is able to activate HSC to synthesize large quantities of ECM, such as various proteoglycans, collagens, and non-collagenous glycoproteins, resulting in an imbalance in the steady state of the ECM in the liver. Therefore, when hepatic fibrosis occurs, TGF-beta 1 will rise synchronously with HA, LN, PC-III, C-IV, reflecting the degree of liver fibrosis.
Compared with a blank control group, the levels of HA, LN, PC-III, C-IV and TGF-beta 1 in the serum of the mice in the model group are obviously increased (P is less than 0.01); compared with the model group, the levels of HA, LN, PC-III, C-IV and TGF-beta 1 in the serum of mice in the ANOLOGICAL FIBRE group and the exendin MIDOI high, medium and low dose groups are all obviously reduced (P is less than 0.01). See table 1.
TABLE 1 fibrosis in sera of groups of mice and TGF-beta1Results of content measurement: (
n＝7，pg/ml)
Note: in comparison with the blank set, the results,*P<0.05，**P<0.01; in comparison with the set of models,△P<0.05，△△P<0.01. the following table is the same.
3.3 liver tissue section staining results of various groups of mice
The normal group of mice has normal hepatocyte structure, normal lobular structure and no fibroplasia; liver cells of mice in the model group have edema, fatty lesion and inflammatory cell infiltration with different degrees, liver lobular structures are damaged, the arrangement is disordered, and fibrous hyperplasia is generated; in the exendin-denscus treatment group, the damage of the liver structure is gradually improved along with the increase of the dosage, the degrees of liver cell edema, fatty lesion and inflammatory cell infiltration are obviously reduced, the fiber deposition, the vascular region or the central venous fiber interval are obviously thinned compared with the model group, and the proliferation of connective tissues is obviously reduced. See fig. 1. Wherein A is a blank group; b is a model group; c is an Anluo chemical fiber group; d is a low-concentration group of the exendin firmus aqueous extract; e is a concentration group in the exendin firmus aqueous extract; f is the group with high concentration of the exendin denseal aqueous extract.
3.4 MMP-13 and TIMP-1 expression in liver tissues of various groups of mice
Compared with a blank control group, the MMP-13 expression in the liver tissues of the mice in the model group is obviously increased (P is less than 0.01), the TIMP-1 expression is obviously reduced (P is less than 0.01), compared with the model group, the MMP-13 expression in the liver tissues of the mice in the ANLOCHEMICAL FIBRE group and the exendin MINII high, medium and low dose groups is obviously increased (P is less than 0.01), and the TIMP-1 expression is obviously reduced (P is less than 0.01). See table 2.
TABLE 2 MMP-13 and TIMP-1 expression in liver tissue of mice in each group: (
n＝6)
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Reference documents:
[1] xu lie ming, liu ping, sheng xi zhong, etc. the combined Chinese and Western medical diagnosis and treatment guideline for liver fibrosis (2019 edition) [ J ]. the journal of combined Chinese and Western medical science, 2019,39(11):1286.
[2] Cheng Xiaoli, Zhao Yu Zhu, Wang Zhiwang, etc. the prevention and treatment effect of the seven-ingredient liver-nourishing granule on hepatic fibrosis rats [ J ] the journal of applied physiology in China 2020,36(05):499 and 502.
[3] Comparison of different concentrations of carbon tetrachloride-induced hepatic fibrosis models in mice [ J ] Experimental animals vs. comparative medicine, 2018,38(04):255 Bian 260.
[4] Chenqi Chinese medicine pharmacology research methodology [ M ] version 2. Beijing, people's health publishing agency, 2006, 1169.
Claims (8)
1. Application of Eremias dens aqueous extract in preparing medicine for treating hepatic fibrosis is provided.
2. Application of exendin media in preparation of drugs for improving MMP-13 expression level in animal liver and simultaneously reducing TIMP-1 expression level in animal liver.
3. The use of claim 1 or 2, wherein the pharmaceutical dosage form comprises an oral formulation.
4. The use of claim 3, wherein the oral formulation comprises a decoction.
5. The use of claim 3, wherein the preparation method of the decoction comprises the following steps:
mixing the exendin brueckii with water, and decocting to obtain the medicine for treating hepatic fibrosis.
6. The use according to claim 5, wherein the mass ratio of the exendin zerumbet to water is 4-5: 100.
7. the use of claim 5, wherein after the decoction, the decoction is further concentrated, and the concentration ratio is (112.5-450): 1000.
8. the use according to claim 5, wherein the temperature of the decoction is 90-100 ℃; the decoction time is 30-40 min.
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