阅读说明：本技术 野蔷薇苷在制备治疗炎性肠病的药物中的应用 (Application of multinoside in preparing medicine for treating inflammatory bowel disease ) 是由 刘艳丽 许琼明 王可昕 于 2021-03-22 设计创作，主要内容包括：本发明公开了一种野蔷薇苷在制备治疗炎性肠病的药物中的应用。本发明采用5％的DSS构建小鼠结肠炎模型评估野蔷薇苷对IBD的治疗作用,提供野蔷薇苷在制备治疗炎性肠病中的应用。发现野蔷薇苷能够治疗结肠炎,且具有不弱于阳性对照药物美沙拉嗪的药效。并且本发明还提供了野蔷薇苷治疗结肠炎是通过诱导DNA修复酶TRAX的水平上升来达到治疗目的的。通过SILAC蛋白质组学的方法,结合质谱分析发现野蔷薇苷可显著上调TRAX蛋白的表达,TRAX在DNA损伤修复中起到了非常关键的作用,可能有助于维持基因组的完整性。进而野蔷薇苷可以通过TRAX蛋白发挥治疗炎症性肠病的作用,修复肠道损伤。(The invention discloses an application of multinoside in preparing a medicament for treating inflammatory bowel disease. The invention adopts 5% DSS to construct a mouse colitis model to evaluate the treatment effect of the multinoside on IBD, and provides the application of the multinoside in preparing medicines for treating inflammatory bowel diseases. The multinoside is found to be capable of treating colitis and has the drug effect which is not weaker than that of the positive control drug mesalazine. The invention also provides a method for treating colitis by using rosapone, which achieves the aim of treatment by inducing the level of DNA repair enzyme TRAX to rise. Through a SILAC proteomics method, the combination with mass spectrometry shows that the multiflorose glycoside can significantly up-regulate the expression of TRAX protein, and the TRAX plays a very critical role in DNA damage repair and can help to maintain the integrity of genome. Furthermore, the multinoside can play a role in treating inflammatory bowel diseases through TRAX protein, and repair intestinal injury.)

1. Application of multinoside in preparing medicine for treating inflammatory bowel disease is provided.

2. The use of claim 1, wherein the inflammatory bowel disease is colitis.

3. The use according to claim 1, wherein the inflammatory bowel disease is proctitis.

4. The use according to claim 1, wherein the inflammatory bowel disease is ileitis.

5. The use according to claim 1, wherein the agent for the treatment of inflammatory bowel disease is an agent that induces an increase in the level of DNA repair enzyme TRAX.

6. The use according to claim 1, wherein the medicament for the treatment of inflammatory bowel disease is administered in a dose of 1 to 100 mg/kg.

7. The use of claim 1, wherein the medicament for treating inflammatory bowel disease is in the form of oral preparation or injection.

Technical Field

The invention relates to the technical field of medicines, in particular to application of multinoside in preparing a medicine for treating inflammatory bowel diseases.

Background

Inflammatory Bowel Disease (IBD) is an idiopathic inflammatory disease of the intestinal tract that affects the ileum, rectum, and colon. The clinical manifestations are diarrhea, abdominal pain and even bloody stool. Inflammatory bowel disease includes Ulcerative Colitis (UC) and Crohn's Disease (CD). Ulcerative colitis is a continuous inflammation of the mucosal and submucosal layers of the colon, and the disease usually involves the rectum and gradually spreads throughout the colon. Crohn's disease affects the whole digestive tract, is a non-continuous, full-thickness inflammation, and is most commonly affected in the terminal ileum, colon and perianal region.

The etiology and pathogenesis of inflammatory bowel disease are not completely clear, and inflammatory response caused by abnormal response of intestinal mucosal immune system is known to play an important role in the pathogenesis of IBD, possibly due to multi-factor interaction, mainly including environmental, genetic, infectious and immune factors.

One of the most threatening risks faced by IBD patients is the risk of colorectal cancer development due to the chronic inflammatory state of the gastrointestinal tract. Literature data indicate that chronic inflammation is the basis for colitis-related colorectal cancer, possibly accounting for 25% of all colorectal tumours diagnosed. During inflammation, the long-term accumulation of neutrophils, macrophages and dendritic cells in the lamina propria and colonic mucosa is accompanied by the release of several cytokines, such as TNF-alpha and IL-1 beta, among others, which induce gene mutations. Subsequently, DNA damage may lead to activation of proto-oncogenes.

Potentilla anserina is an enlarged root tuber of Potentilla anserina of Rosaceae, also called "ginseng fruit", and is commonly used in Tibetan folks to treat malnutrition, weakness of the spleen and stomach, diarrhea, etc. The multinoside is a compound with higher content in small molecules of the silverweed cinquefoil root, and is one of representative active ingredients of the silverweed cinquefoil root. The multinoside has pharmacological activities of oxidation resistance, virus resistance, inflammation resistance and the like: if the rat is in acute hypoxia state for a long time, the oxidative stress and inflammation can be enhanced, and the high dose of the rosa multiflora glycosides can relieve the bone injury of the rat caused by the acute hypoxia state; in the research of the effect of the rosapons on myocardial cell oxidative stress injury, the rosapons can increase Bcl-2 by reducing the levels of Bax, Cyt-c, Caspase-9 and Caspase-3, thereby protecting H9c2 myocardial cells from H₂O₂Inducing oxidative stress and apoptosis, reducing the production of ROS and MDA, inhibiting calcium overload, and improving antioxidant enzyme activity, and multinoside can be used for preventing and treating H by its antioxidant and anti-apoptosis effects₂O₂Potential for induced oxidative stress damage of cardiomyocytes. The previous period of the subject group also adopts the rosa-multiflora glycoside to protect the small intestine from being damaged by irradiation, mainly through oxidative stress, and the previous literature also relates to the expression that the effect is achieved through the oxidative stress. At present, no research report of multinoside on the aspect of DNA damage repair exists.

Disclosure of Invention

In order to solve the technical problems, the invention adopts 5% DSS to construct a mouse colitis model to evaluate the therapeutic effect of the rosaniline on IBD, and provides the application of the rosaniline in preparing medicines for treating inflammatory bowel diseases.

The first purpose of the invention is to provide application of multinoside in preparing a medicament for treating inflammatory bowel diseases.

Further, the inflammatory bowel disease is colitis.

Further, the inflammatory bowel disease is proctitis.

Further, the inflammatory bowel disease is ileitis.

Further, the drug for treating inflammatory bowel disease is a drug that induces an increase in the level of DNA repair enzyme TRAX.

Further, the administration dose of the medicament for treating the inflammatory bowel disease is 1-100 mg/kg.

Furthermore, the dosage form of the medicament for treating the inflammatory bowel disease is oral preparation or injection.

By the scheme, the invention at least has the following advantages:

the invention discovers that the multinoside can treat colitis and has the drug effect which is not weaker than that of a positive control drug mesalazine. The invention also provides a method for treating colitis by using rosapone, which achieves the aim of treatment by inducing the level of DNA repair enzyme TRAX to rise. Through a SILAC proteomics method, the combination with mass spectrometry shows that the multiflorose glycoside can significantly up-regulate the expression of TRAX protein, and the TRAX plays a very critical role in DNA damage repair and can help to maintain the integrity of genome. Furthermore, the multinoside can play a role in treating inflammatory bowel diseases through TRAX protein, and repair intestinal injury.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

FIG. 1 is a graph of the effect of multinoside on the weight score of mice with DSS-induced colitis; the multinoside is administered by gavage at dosage of 6mg/kg and 12mg/kg for 9 days, 1 time per day; continuously intragastrically administering mesalazine with a dose of 400mg/kg for 9 days, inducing colitis of the mice by using 5% DSS 24 hours after administration, continuously administering for 7 days, and killing the mice on the 8 th day after the colitis is induced; calculating the body weight scores of all groups of mice according to the scoring requirements every day;

FIG. 2 is a graph of the effect of multinoside on the evaluation of stool characteristics of mice with DSS-induced colitis; the multinoside is administered by gavage at dosage of 6mg/kg and 12mg/kg for 9 days, 1 time per day; continuously intragastrically administering mesalazine with a dose of 400mg/kg for 9 days, inducing colitis of the mice by using 5% DSS 24 hours after administration, continuously administering for 7 days, and killing the mice on the 8 th day after the colitis is induced; calculating the fecal character scores of all groups of mice every day according to the scoring requirements;

FIG. 3 is a graph of the effect of multinoside on the bloody stool score of mice with DSS-induced colitis; the multinoside is administered by gavage at dosage of 6mg/kg and 12mg/kg for 9 days, 1 time per day; continuously intragastrically administering mesalazine with a dose of 400mg/kg for 9 days, inducing colitis of the mice by using 5% DSS 24 hours after administration, continuously administering for 7 days, and killing the mice on the 8 th day after the colitis is induced; calculating the bloody stool score of each group of mice according to the scoring requirement every day;

FIG. 4 is a graph of the effect of multinoside on DAI scores in mice with DSS-induced colitis; the multinoside is administered by gavage at dosage of 6mg/kg and 12mg/kg for 9 days, 1 time per day; continuously intragastrically administering mesalazine with a dose of 400mg/kg for 9 days, inducing colitis of the mice by using 5% DSS 24 hours after administration, continuously administering for 7 days, and killing the mice on the 8 th day after the colitis is induced; calculating DAI scores of all groups of mice according to the scoring requirements every day;

FIG. 5 is a graph of the effect of multinoside on colon length in DSS-induced colitis mice; measuring the colon length of each group of mice after the mice are sacrificed;

FIG. 6 is a graph of the effect of multinoside on spleen weight in mice with DSS-induced colitis; weighing the spleen of each group of mice after sacrifice;

FIG. 7 is a graph of the effect of multinoside on spleen index in mice with DSS-induced colitis; weighing the spleen of each group of mice after the mice are sacrificed, and calculating the spleen index of each group of mice;

figure 8 is a proteomic volcano plot.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1:

constructing a mouse colitis model: ICR mice were first fed normally for 5 days with free water diet during the feeding period. The mice were randomly divided into groups after weighing, including a normal control group, a DSS model group, a multinoside (6, 12mg/kg) group, and a positive drug mesalazine (400mg/kg) group. Feeding dog rose glycoside and mesalazine into stomach, dissolving DSS in drinking water of mice 24h after feeding, continuously feeding 5% DSS7 days to model group and feeding group mice, weighing body weight every day, observing stool characters of the mice, checking occult blood/hematochezia conditions of the mice, calculating DAI score, killing the mice after feeding on day 8, and taking blood, colon and spleen. The length of the colon is measured, and the weight of the colon and the spleen is measured.

Example 2:

a5% DSS is adopted to construct a mouse colitis model to evaluate the treatment effect of the multinoside on IBD, and the mice are continuously given 5% DSS7 days, so that the results show that the mice are listened by DSS molding for 3 days, the feces are in a thin and soft state and are in a non-forming state, and occult blood or hematochezia conditions exist, and the mice show watery feces and serious hematochezia on the 6 th day of molding. Statistical experimental data show that the multiflora rose glycoside 12mg/kg group can obviously reduce the fecal score of mice from the 4 th day, has significant difference with a model group (P <0.001), and has equivalent fecal character recovery effect with the mesalazine group mice (figure 2). Also as shown in fig. 3, the 12mg/kg multiflora glycoside group significantly reduced the stool score of mice from day four and was statistically significantly different (P <0.01) compared to the model group mice. The DAI scores of the mice are comprehensively evaluated, from the 3 rd day of model building, the colitis of the mice of the DSS model building group is aggravated, the DAI scores are increased and reach a peak at the 6 th day, and the DAI scores of the mice are remarkably reduced after the 4 th day of giving 12mg/kg of multiflora rose glycoside (figure 4), and have remarkable difference (P <0.001) with the mice of the model group. The above experimental results show that the group with a dose of 12mg/kg of multiflora rose glycoside can relieve the symptoms of colitis in mice.

The colon length of the mice is counted, and the results are shown in figure 5, and the colon length of the mice of the DSS model group is significantly shortened compared with that of the normal control group (P < 0.001). The multinoside 12mg/kg dose group and the mesalazine group both recovered the colon length of mice, and the effect was equivalent, and was significantly different compared with the model group (P < 0.05).

After weighing the body weight and spleen weight of the mice, the spleen weight of different groups of mice was counted and the spleen index was calculated according to the formula [ spleen index ═ spleen weight/body weight ]. The results are shown in fig. 6 and fig. 7, and the spleen weight of the mice in the DSS model group is significantly increased compared with the normal control group, with significant difference (P < 0.001). The dog rose glycoside 12mg/kg group and the mesalazine group can obviously reduce the spleen weight of mice after administration, can recover to a normal level, have very significant statistical difference (P <0.001) compared with the mice in a model group, and the drug effect of the dog rose glycoside 12mg/kg group is equivalent to that of the mesalazine group.

Example 3:

we grown and passaged Caco2 colon cells in SILAC medium for at least five passages until more than 95% of the protein was labeled. Then, the cells were inoculated on a 10cm plate, cultured for 24 hours, and then multinoside (50. mu.M) or DMSO was added to the cells cultured in the "heavy" or "light" medium for further culture for 1 hour. Cells cultured in either "heavy" or "light" medium were then treated with the DNA damaging agent cisplatin (20. mu.M) for 4 h. The cells were lysed with 8M urea buffer to obtain total proteins, and total protein samples were prepared for the model group and the rosaniline-administered group, respectively. Samples were analyzed on an Orbitrap Fusion Lumos Mass spectrometer (Thermo Fisher Scientific), data processed by Max Quant software, and proteins with FDR ≦ 0.01 were considered positive. The P-value of the identified protein was calculated using the IBM SPSS software (Ver19) independent sample t-test function. Proteins with fold changes >1.50 or <0.67 and P <0.05 were considered significantly differentially expressed proteins.

We identified a total of 4661 proteins, with a quantitation of 2184 proteins, the proportion of which is shown in the volcano plot (figure 8). In the quantified proteins, eight proteomes with fold changes >1.50 or <0.67 and P <0.05 were labeled with red (up-regulated) or green (down-regulated) spots. Compared with the model group, the expression of TRAX is up-regulated by more than 11 times after administration of multinoside, which is the most changed protein. TRAX is a multifunctional protein that can participate in different signaling pathways through interactions with multiple proteins to exert lesion repair effects. In conclusion, the rosapons are thought to play a role in treating inflammatory bowel diseases through TRAX protein, so as to repair intestinal injury.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.