Rat enterotoxicity model construction method based on dysosma versipellis

Abstract

The invention provides a method for constructing a rat enterotoxicity model based on dysosma versipellis, and belongs to the technical field of animal models. The construction method of the rat enterotoxicity model comprises the following specific steps: 1) preparing a dysosma versipellis alcohol extract and a healthy rat; 2) carrying out gavage treatment on the healthy rats by using the dysosma versipellis alcohol extract; the dosage of the dysosma versipellis alcohol extract in the gavage treatment is 1.2 g / kg / d, and the gavage time is 3 days; and 3) after the rat is subjected to gavage treatment, judging whether the rat enterotoxicity model is successfully constructed according to the detection results of comprehensive toxicity expression, organ index change, histopathology and serum biochemical indexes of the rat. The method is simple in step and scientific and reasonable in evaluation index, the dysosma versipellis-based rat enterotoxicity model is successfully prepared, a theoretical basis is further provided for use of dysosma versipellis, and the blank of research in related fields is filled.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models, and particularly relates to a method for constructing a rat intestinal toxicity model based on Dysosma versipellis. Background Art

[0002] Dysosma versipellis (Hance) M. Cheng is a perennial herb of the genus Dysosma in the Berberidaceae family. It is bitter in taste and cool in nature, and its rhizome can be used as medicine. It is mainly used to resolve phlegm and dissipate nodules, dispel stasis and relieve pain, clear heat and detoxify, and treat scrofula and goiter. The chemical components in Dysosma versipellis include vanillic acid, quercetin, kaempferol, podophyllotoxin, podophyllotoxone, diphyllin, 4'-demethylpodophyllotoxin, dehydro-podophyllotoxin, deoxypodophyllotoxin, etc. It can be obtained from the literature that the lignin components in Dysosma versipellis have obvious pharmacological effects, but are highly toxic, and there have been multiple poisoning cases in recent clinical applications. Since the pharmacological effects of Dysosma versipellis are closely related to its toxic components, there is currently a lack of a rat intestinal toxicity model of Dysosma versipellis, as well as a method for constructing a rat model of the intestinal toxicity model of Dysosma versipellis and evaluation indicators.

[0003] Therefore, there is an urgent need for a method for constructing a rat intestinal toxicity model based on Dysosma versipellis with a simple construction method and reasonable evaluation indicators. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for constructing a rat intestinal toxicity model based on Dysosma versipellis. The method of the present invention has simple steps, and the evaluation indicators are scientific and reasonable, and a rat intestinal toxicity model based on Dysosma versipellis is successfully prepared.

[0005] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a method for constructing a rat intestinal toxicity model based on Dysosma versipellis, comprising the following steps:

[0007] 1) Prepare an ethanol extract of Dysosma versipellis and healthy rats;

[0008] 2) Perform gavage treatment on the healthy rats with the ethanol extract of Dysosma versipellis; the dosage of the ethanol extract of Dysosma versipellis in the gavage treatment is 1.2 g / kg / d, and the gavage time is 3 days;

[0009] 3) After the gavage treatment of the rats, judge whether the construction of the rat intestinal toxicity model is successful according to the comprehensive toxicity performance of the rats, changes in organ indices, histopathology, and serum biochemical index detection results.

[0010] Preferably, the ethanol extract of Dysosma versipellis is obtained by extracting the roots and / or stems of the plant Dysosma versipellis in the Berberidaceae family.

[0011] Preferably, the healthy rats are SPF - grade male SD rats with a body weight of 250±20g.

[0012] Preferably, the comprehensive toxicity manifestations include a significant decrease in body weight, and phenomena such as rough hair, arched back, piloerection, unclean anus, loose stools, etc. In a small number of cases, there are nose and mouth bleeding, eye bleeding, and ecchymosis at the joints of the forelimbs.

[0013] Preferably, the changes in organ indices include changes in intestinal weight.

[0014] Preferably, the tissue sites for histopathology include the duodenum, ileum, and colon.

[0015] Preferably, the duodenal lesions include obvious dilation of the subepithelial space of the mucosal layer of some intestinal villi, a decrease in the number of goblet cells, a small amount of inflammatory cell infiltration, a small amount of cell necrosis, and karyopyknosis and deep staining of the cell nuclei;

[0016] The ileal lesions include the presence of Gruenhagen's space under the mucosal layer of some intestinal villi, accompanied by capillary congestion, a small amount of inflammatory cell infiltration in the tissue, a small amount of cell necrosis, and karyopyknosis and deep staining of the cell nuclei;

[0017] The colonic lesions include erosion and shedding of epithelial cells in the mucosal layer of some parts, exposure of the lamina propria, a decrease in the number of goblet cells, no edema in the submucosa, and a small amount of inflammatory cell infiltration in the tissue.

[0018] Preferably, the serum biochemical indices include Gas, MDA, IL - 1β, AMS, and Pepsin.

[0019] It has at least the following beneficial technical effects:

[0020] The present invention provides a method for constructing a rat intestinal toxicity model based on Dysosma versipellis. The healthy rats are treated by gavage with an ethanol extract of Dysosma versipellis. The dosage of the ethanol extract of Dysosma versipellis in the gavage treatment is 1.2g / kg / d, and the gavage time is 3 days. Whether the model is successfully established is judged by the comprehensive toxicity manifestations, changes in organ indices, histopathology, and detection results of serum biochemical indices. This method is simple, has a fast modeling speed, and the obtained model is scientific and reasonable. Description of the Drawings

[0021] Figure 1 It is a graph of the toxicity manifestations of rats.

[0022] Figure 2 It is a graph of the influence of serum biochemical indices of rats.

[0023] Figure 3 It is a histological section graph of the intestine. Detailed Embodiments

[0024] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0028] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0029] As used in the present invention, "room temperature" and "normal temperature" are both calculated as 25 ± 2 °C unless otherwise specified.

[0030] Unless otherwise specified, the raw materials or instruments used in the following examples of the present invention are all obtained commercially.

[0031] Example 1

[0032] 1 Experimental Materials

[0033] 1.1 Medicinal Herbs and Reagents

[0034] Dysosma versipellis (from Anxing Chinese Herbal Medicine Slices Co., Ltd., Anguo City, Hebei Province), identified by Professor Yang Yaojun of Beijing University of Chinese Medicine as the roots and rhizomes of the plant Dysosma versipellis in the Berberidaceae family.

[0035] Pentobarbital sodium (sub-packaged by Shanghai Chemical Reagent Procurement and Supply Station).

[0036] Analytical grade ethanol (Shanghai Yien Chemical Reagent Co., Ltd.).

[0037] 1.2 Instruments

[0038] Low-temperature high-speed centrifuge (Beijing Times Beili Centrifuge Co., Ltd.); Rotary evaporator (Shanghai Ailang Instruments Co., Ltd.); KQ-300DB type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); Pipette (Eppendorf, Germany).

[0039] 1.3 Animals

[0040] Thirty-two SPF-grade male SD rats, purchased from Spf (Beijing) Biotechnology Co., Ltd., with a body weight of about 250 ± 20 g. After the rats were purchased, they were raised under controlled environmental conditions with an environmental temperature of 23 ± 2 °C and an environmental humidity of 35 ± 5%. The study has obtained the approval of the Experimental Animal Ethics Review Committee of Beijing University of Chinese Medicine (approval number: BUCM-4-2022112101-4060) and strictly complied with the relevant guidelines.

[0041] 2 Experimental methods

[0042] 2.1 Preparation of Dysosma versipellis ethanol extract and anesthetic

[0043] 2.1.1 Preparation of Dysosma versipellis ethanol extract

[0044] Take 30 g of small pieces of Dysosma versipellis, and perform two reflux extraction operations. For the first time, add 10 times the amount of 50% ethanol and reflux for 1.5 h. For the second time, add 8 times the amount of 50% ethanol and reflux for 1.5 h. Filter, combine the two filtrates, and rotary evaporate (reduce the pressure to concentrate) to obtain an extract. Dissolve it with distilled water and then dilute it to a test drug of 0.15 g / mL for standby.

[0045] 2.1.2 Preparation of anesthetic

[0046] Weigh an appropriate amount of sodium pentobarbital with a balance, then pour it into a beaker, then add the required deionized water, and stir well with a magnetic stirrer for 5 - 10 min to prepare a 2% sodium pentobarbital solution. The anesthetic dose for intraperitoneal injection in rats is: 50 mg / kg.

[0047] 2.2 Animal grouping

[0048] After the rats were purchased, they were raised under controlled environmental conditions with an environmental temperature of 23 ± 2 °C and an environmental humidity of 35 ± 5%. The rats were randomly divided into 2 groups, with 16 rats in each group. They were the blank control group (Normal Saline, NS) and the drug administration group (Dysosma versipellis (Hance) M.Cheng, DV).

[0049] 2.3 Administration and Sample Collection

[0050] The rats in the DV group were intragastrically administered with 1.2 g / kg / d of the ethanol extract of Dysosma versipellis for 3 consecutive days, and the rats in the NS group were given the same volume of distilled water. The activity status of the mice was observed every day, and the weights of the mice in each group were recorded before and after administration. The experimental design is shown in Table 1.

[0051] Table 1 Experimental Design of Dysosma versipellis Administration

[0052]

[0053]

[0054] 2.4 Toxic Manifestations in Animals

[0055] The body weights were measured before each administration for 3 consecutive days of drug administration. A reference table for the toxic phenotypes of experimental animals and a Bristol stool scale were formulated, and based on this, the phenotypic changes such as the appearance, body weight, and excretion status of the rats were observed and recorded.

[0056] 2.5 Tissue Collection and Processing

[0057] 8 mL of abdominal aortic blood was taken and placed in a non-heparinized test tube. The sample was centrifuged at 4 °C and 3000 r / min for 15 min, and the supernatant was collected.

[0058] The supernatant was taken out of the test tube and centrifuged again at 4 °C and 3500 r / min for 8 min to further purify the sample. Finally, the supernatant was stored in a -80 °C refrigerator for subsequent analysis.

[0059] After drug administration and blood collection, the rats in each group were sacrificed, and the intestinal tissues of the control group and the Dysosma versipellis administration group were quickly taken out. The abdominal cavity of the mice was opened (photographed), the whole stomach of the mice was removed, photographed on grid paper, and cut in half along the greater curvature of the stomach from the cardia. Half was fixed with 4% paraformaldehyde solution and stored at room temperature for histopathological examination; the other half was quickly frozen with liquid nitrogen.

[0060] Collect the colon tissue at 0 - 3 cm behind the cecum, the ileum tissue at 0 - 1 cm in front of the cecum, and the duodenum tissue at 0 - 3 cm in the lower part of the stomach of mice. Wash them clean with physiological saline and strip the connective tissue, then fix them with 4% paraformaldehyde (10 pieces, and the remaining ones are cryopreserved in an -80 °C refrigerator); then collect the colon tissue at 3 - 6 cm behind the cecum, the ileum tissue at 1 - 2 cm in front of the cecum, and the duodenum tissue at 3 - 6 cm in the lower part of the stomach. After cutting the tissue into appropriate sizes, quickly and completely immerse it in 5 - 10 times the volume of RNAsolidTM reagent. Collect the colon tissue at 6 - 10 cm behind the cecum, the ileum tissue at 2 - 4 cm in front of the cecum, and the duodenum tissue at 6 - 10 cm in the lower part of the stomach. Wash them clean with physiological saline and strip the connective tissue. After cutting the tissue into appropriate sizes, quickly and completely immerse it in 5 - 10 times the volume of RNAsolid tissue RNA stable preservation solution. The samples stored in RNAsolid tissue RNA stable preservation solution are soaked overnight at 4 °C. Aspirate and discard the RNAsolid protection solution, and then directly put the samples into -80 °C.

[0061] 2.6 Organ index of target organs

[0062] After removing the contents of the gastrointestinal tissues of rats, weigh them and calculate the organ index: Organ index = Organ weight / Rat body weight × 100%, and place them in liquid nitrogen and store at -80 °C.

[0063] 2.7 Determination of serum indexes of intestinal tissues

[0064] Determine the contents or activities of interleukin - 1 (IL - 1β), interleukin - 6 (IL - 6), interleukin - 10 (IL - 10), malondialdehyde (MDA), amylase (AMS), serum diamine oxidase (DAO), reactive oxygen species (ROS), pepsin, gastrin (Gas), motilin (MTL), tumor necrosis factor α (TNF - α), and gastric mucosal transforming growth factor α (α - TGF) in the supernatant of tissue homogenate. Use HE staining for pathological tissues to observe the tissue damage of animals after drug exposure.

[0065] Specific operation: After paraffin sectioning, dewax with xylene, hydrate, stain with hematoxylin for 10 min, differentiate, stain with eosin, dehydrate, make transparent with xylene, and seal with neutral resin glue. Detect and histologically evaluate the pathological changes of rat intestines through an optical microscope.

[0066] 2.8 Data processing

[0067] Statistical analysis of the obtained data was performed using GraphPad Prism software. First, the data were tested for normality. If the data were normally distributed, an independent samples t-test was used to compare between groups; otherwise, non-parametric tests were employed. The significance level was set at α = 0.05. If P < 0.05, the results were considered statistically significant.

[0068] 3 Results

[0069] 3.1 Animal toxicity manifestations

[0070] After continuous administration for 3 days, compared with the NS group, the body weights of the DV group were significantly reduced, and phenomena such as rough hair, arched back, piloerection, unclean anus, and loose stools occurred. In a small number of cases, phenomena such as bleeding from the nose and mouth, eye bleeding, and ecchymosis at the joints of the front limbs occurred. The specific results are shown in Figure 1 。

[0071] 3.2 Changes in organ indices

[0072] The calculation results of the organ indices of rat livers showed that compared with the blank group, the intestinal weights of the experimental group decreased, as shown in Table 4.

[0073] Table 4 Changes in the average organ indices of rats

[0074]

[0075] 3.3 Serum biochemical indices

[0076] The specific effects of Dysosma versipellis on the serum biochemical indices of rats in the NS group and the DV group are shown in Figure 2 。It can be seen from the charts that compared with the NS group, the levels of Gas, MDA, IL-1β, and AMS in the DV group increased, and the level of Pepsin decreased, and all were statistically significant (P < 0.01).

[0077] 3.4 Histopathology

[0078] See Figure 3 ,where A1, A2, and A3 are the NS group; B1, B2, and B3 are the DV group.

[0079] NS group, duodenum: The intestinal tissue structure was basically normal. The intestinal villi structure was clear in the field of view, and the epithelial cells were arranged neatly and tightly without shedding. Ileum: The intestinal tissue structure was basically normal. The epithelial cells of the mucosal layer were arranged neatly and tightly without shedding in the field of view; the crypt structure was intact and arranged neatly; there was no edema in the submucosa; Colon: The intestinal tissue structure was basically normal. The intestinal villi structure was clear in the field of view, and the epithelial cells of the mucosal layer were arranged neatly and tightly without shedding; goblet cells were abundant; the crypt structure was intact and arranged neatly. No obvious inflammatory cell infiltration was seen in the intestinal tissue.

[0080] DV group, duodenum: Moderate abnormality in intestinal tissue structure, as indicated by the red arrow. The submucosal space of the epithelial layer of some intestinal villi in the field of view is significantly dilated; goblet cells, reduced in number; a small amount of inflammatory cell infiltration and a small amount of cell necrosis with karyopyknosis and deep staining can be seen in the tissue. Ileum: Moderate abnormality in intestinal tissue structure, as indicated by the red arrow. Gruenhagen's space can be seen under the epithelial layer of some intestinal villi in the field of view, accompanied by capillary congestion; as indicated by the yellow arrow, a small amount of inflammatory cell infiltration can be seen in the tissue. As indicated by the black arrow, a small amount of cell necrosis with karyopyknosis and deep staining. Colon: Mild abnormality in intestinal tissue structure, as indicated by the red arrow. Erosion and exfoliation of epithelial cells in some mucosal layers in the field of view, with the lamina propria exposed; goblet cells, reduced in number; no edema is seen in the submucosa; a small amount of inflammatory cell infiltration can be seen in the tissue.

[0081] 4 Discussion

[0082] To evaluate the intestinal toxicity of Dysosma versipellis extract in SD rats, the rats were intragastrically administered with 1.2 g / kg / d of the ethanol extract of Dysosma versipellis for 3 consecutive days. Evaluation was conducted by observing the toxic symptoms of the experimental animals, analyzing the pathological sections of intestinal tissues, and detecting the corresponding blood biochemical indexes. During the experimental period, the rats in the DV group gradually had a reduced food intake. Compared with the NS group, their body weights were significantly decreased, and they showed phenomena such as rough hair, varying degrees of dehydration, unclean anus, loose stools, laziness in movement, arched back, erected hair, and deepened breathing. Most of them had ecchymosis in the forelimb joints and bleeding from the nose and mouth, and a small number had tremors and eye bleeding. Malondialdehyde (MDA) is one of the commonly used indicators to measure the degree of oxidative stress. It is the final oxidation product produced by free radicals acting on lipids, which can cause cross-linking and polymerization of biological macromolecules such as nucleic acids and proteins and has cytotoxic effects. After the administration of Dysosma versipellis, the MDA level increased significantly (P<0.001). Interleukin-1 (IL-1β) is a protein with immunomodulatory effects, especially capable of regulating the acute-phase response. It can cause leukocyte aggregation and stimulate the expression of intercellular adhesion molecules by endothelial cells and leukocytes, thereby mediating neutrophil infiltration and aggravating the inflammatory response in the damaged area. The level in the DV group was significantly higher than that in the NS group (P<0.01). Serum gastrin (GAS) plays an important role in regulating gastrointestinal function. It can not only promote the secretion of gastric acid and pepsin, but also promote gastric mucosa regeneration, improve gastrointestinal motility, and accelerate gastric emptying. The experimental results showed that compared with the NS group, the GAS level in the DV group increased significantly (P<0.001). As a pathological manifestation of intestinal injury, the duodenum showed obvious dilation of the subepithelial space of the intestinal villus mucosal layer and a decrease in the number of goblet cells; a small amount of inflammatory cell infiltration and a small amount of cell necrosis were visible in the tissue, with deeply stained and pycnotic nuclei. The ileum toxicity manifested as the presence of Gruenhagen's space under the intestinal villus mucosal layer, accompanied by capillary congestion, a rich number of goblet cells, a small amount of inflammatory cell infiltration, and a small amount of cell necrosis, with deeply stained and pycnotic nuclei. The colon toxicity manifested as erosion and exfoliation of the epithelial cells of the mucosal layer, exposure of the lamina propria, a decrease in the number of goblet cells, and a small amount of inflammatory cell infiltration in the tissue. In this experiment, different degrees of damage were observed in the intestinal tissues of rats after 3 days of administration of the ethanol extract of Dysosma versipellis through histopathological examination, indicating that obvious lesions occurred in the intestines of the rats in the administration group. Based on the comprehensive toxic phenotypes, histopathological examination, and serum biochemical index detection results, it can be concluded that after 3 days of administration of 1.2 g / kg / d of the ethanol extract of Dysosma versipellis, obvious toxic manifestations occurred in the rats' intestines, that is, the model was successfully established.

[0083] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing a rat intestinal toxicity model based on Dysosma versipellis, characterized in that, It includes the following steps: 1) Prepare the ethanol extract of Dysosma versipellis and healthy rats; 2) Administer the ethanol extract of Dysosma versipellis to the healthy rats by gavage; the dosage of the ethanol extract of Dysosma versipellis in the gavage treatment is 1.2 g / kg / d, and the gavage time is 3 days; 3) After the rats are treated by gavage, judge whether the intestinal toxicity model of the rats is successfully constructed according to the comprehensive toxicity performance, changes in organ indices, histopathology and serum biochemical index detection results of the rats.

2. The method for constructing a rat intestinal toxicity model according to claim 1, characterized in that, The ethanol extract of Dysosma versipellis is obtained by extracting the roots and / or stems of the plant Dysosma versipellis of the Berberidaceae family.

3. The method for constructing a rat intestinal toxicity model according to claim 1, wherein The healthy rats are SPF-grade male SD rats with a body weight of 250±20 g.

4. The method for constructing a rat intestinal toxicity model according to claim 1, characterized in that The comprehensive toxicity performance is that the body weights are all significantly reduced, and phenomena such as rough hair, arched back, erected hair, unclean anus, loose stools, etc. occur, and a small number show nose and mouth bleeding, eye bleeding, and ecchymosis at the joints of the forelimbs.

5. The method for constructing a rat intestinal toxicity model according to claim 1, wherein The changes in organ indices include changes in intestinal weight.

6. The method for constructing a rat intestinal toxicity model according to claim 1, characterized in that, The tissue sites of the histopathology include the duodenum, ileum, and colon.

7. The method for constructing a rat intestinal toxicity model according to claim 6, characterized in that, The duodenal lesions include obvious dilation of the subepithelial space of the mucosa layer of some intestinal villi, a decrease in the number of goblet cells, a small amount of inflammatory cell infiltration and a small amount of cell necrosis can be seen, and the cell nuclei are pyknosis and deeply stained; The ileal lesions include the presence of Gruenhagen’s space under the mucosa layer of some intestinal villi, accompanied by capillary congestion, a small amount of inflammatory cell infiltration in the tissue, a small amount of cell necrosis, and the cell nuclei are pyknosis and deeply stained; The colonic lesions include erosion and shedding of epithelial cells in the mucosa layer of some parts, exposure of the lamina propria, goblet cells, a decrease in the number, no edema in the submucosa layer, and a small amount of inflammatory cell infiltration in the tissue.

8. The method for constructing a rat intestinal toxicity model according to claim 1, wherein The serum biochemical indices include Gas, MDA, IL-1β, AMS, and Pepsin.

Images