阅读说明：本技术 绿原酸化合物在制备治疗由don引起炎症的药物中应用 (Application of chlorogenic acid compound in preparing medicine for treating inflammation caused by DON ) 是由 尹清强 许小向 常娟 王平 卢富山 王潇 刘超齐 宋安东 李茂龙 李慧娟 耿启泉 于 2019-12-03 设计创作，主要内容包括：本发明实施例公开了式I所示的绿原酸化合物及其药学上可接受的盐类,在制备治疗由DON引起的炎症的药物中应用。本发明实施例的绿原酸对呕吐毒素诱导猪肠道上皮细胞炎症和凋亡的保护作用。随着DON浓度的增加及培养时间的延长,细胞活力显著降低。CGA可以显著增加细胞活力、减少乳酸脱氢酶(LDH)释放和细胞凋亡率。此外,CGA还可以显著下调由DON诱导的猪肠道上皮细胞(IPEC-J2)中IL-6、IL-8、TNF-α、COX-2、Bax、Caspase 3和ASCT2基因的相对表达量,并显著上调由DON诱导的IPEC-J2细胞中ASCT2、ZO-1、Occludin、Claudin-1、PePT1和GLUT2基因的表达。本发明实施例的CGA可以减轻DON诱导IPEC-J2细胞的细胞毒性、炎症和凋亡,保护肠道的完整性和动物的健康。(The embodiment of the invention discloses application of a chlorogenic acid compound shown in a formula I and pharmaceutically acceptable salts thereof in preparing a medicament for treating inflammation caused by DON, wherein the chlorogenic acid compound has a protective effect on vomitoxin-induced porcine intestinal epithelial cell inflammation and apoptosis, and the cell viability is remarkably reduced along with the increase of DON concentration and the extension of culture time.)

1. Chlorogenic acid compound shown as formula I and pharmaceutically acceptable salts thereof, and application of chlorogenic acid compound in preparation of medicines for treating inflammation caused by DON

2. The use according to claim 1,

the inflammation is DON-induced intestinal epithelial cell inflammation.

3. The use according to claim 2,

the chlorogenic acid compound is used for up-regulating the expression abundance of IL-8 gene mRNA in IPEC-J2 cells induced by DON and down-regulating the expression abundance of TNF- α gene mRNA.

4. The use according to claim 3,

the chlorogenic acid compound is used for down-regulating expression of mRNA abundance of a Bcl-2 gene in the IPEC-J2 cells induced by DON and up-regulating mRNA abundance of the claudin in the IPEC-J2 cells induced by DON.

5. The use according to claim 3,

the chlorogenic acid compound is used for up-regulating the abundance of PepT1 in IPEC-J2 cells induced by DON and promoting the up-regulation of the abundance of glucose transporter mRNA.

6. The use according to any one of claims 1 to 5, wherein the chlorogenic acid compound or the pharmaceutically acceptable salt thereof comprises an effective amount of a pharmaceutical composition comprising one or more pharmaceutically acceptable pharmaceutical carriers.

7. The use of claim 6, wherein the drug carrier comprises:

diluent, excipient, adhesive, wetting agent, absorption enhancer, surfactant and lubricant.

8. The use of claim 6, wherein the pharmaceutical composition is in the form of a tablet, capsule, oral liquid, injection, powder or paste.

Technical Field

The embodiment of the invention relates to the technical field of biological medicines, and particularly relates to application of chlorogenic acid in preparing a medicine for treating inflammation caused by DON.

Background

Deoxynivalenol (DON), also known as emetic toxin, is a type B trisporatoxin produced by fungi such as Fusarium graminearum and Fusarium culmorum. Vomitoxin is commonly present in grains and feed crops such as wheat, corn, oat, barley, meals, lees and the like all over the world, can generate wide toxic effect after being eaten by animals, and has potential threat to the health of human beings and animals. It has been reported that DON and its derivatives may be transmitted to the human body by eating contaminated food and water, inhalation, or skin contact. It was found that residual DON and its metabolites were also detected in pigs fed diets containing different levels of DON. When animals are exposed to DON for extended periods of time, they may cause vomiting, food refusal, decreased food intake, stunting, diarrhea, immune system disorders, neurological disorders, and ultimately death.

Among the common livestock, swine is the animal model most relevant to human sensitivity, and is most sensitive to DON. The intestinal tract is an organ integrating functions of digestion, absorption, endocrine, immunity and defense, and is easily damaged by harmful substances such as toxin, bacteria, anti-nutritional factors and the like. Furthermore, the gut is the primary target of DON and various intestinal diseases may occur. Diarrhea is a common intestinal disease that can cause about 5% of the deaths of weaned piglets per year. It has been reported that DON can induce apoptosis and inflammation in IPEC-J2 cells by increasing expression of genes associated with inflammation and apoptosis. Therefore, it is essential to find an effective substance to reduce inflammation and apoptosis caused by DON to maintain intestinal health.

Phenolic acids have important roles in defense against plant fungal pathogens and in inhibiting the production of various mycotoxins in vitro. Chlorogenic acid (CGA) is a phenylpropanoid analogue synthesized by photosynthesis of plants, and is a main active ingredient of traditional Chinese medicinal materials such as eucommia ulmoides, honeysuckle and the like.

In summary, how to develop a drug for alleviating or treating the intestinal inflammation induced by DON is a technical problem to be solved urgently.

Disclosure of Invention

Therefore, the embodiment of the invention provides application of chlorogenic acid compounds in preparing a medicament for treating inflammation caused by DON, so as to solve the problem that the prior art lacks a medicament for effectively relieving or treating intestinal inflammation induced by DON.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

chlorogenic acid compound shown as formula I and pharmaceutically acceptable salts thereof are applied to preparation of medicines for treating inflammation caused by DON.

Preferably, the inflammation is DON-induced inflammation of the intestinal epithelium.

Preferably, the chlorogenic acid compound is used for up-regulating the expression abundance of IL-8 gene mRNA in IPEC-J2 cells induced by DON and down-regulating the expression abundance of TNF- α gene mRNA.

Preferably, the chlorogenic acid compounds are used for down-regulating expression of mRNA abundance of the Bcl-2 gene in DON-induced IPEC-J2 cells and up-regulating mRNA abundance of the claudin in DON-induced IPEC-J2 cells.

Preferably, the chlorogenic acid compounds are used to up-regulate DON-induced PepT1 abundance in IPEC-J2 cells and facilitate up-regulation of glucose transporter mRNA abundance.

Preferably, the pharmaceutical composition comprises an effective dose of the chlorogenic acid compound or the pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable pharmaceutical carriers.

Preferably, diluents, excipients, binders, wetting agents, absorption enhancers, surfactants, lubricants.

Preferably, the dosage form of the pharmaceutical composition is tablets, capsules, oral liquid, injection, powder or paste.

In the embodiment of the present invention, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, and the like. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

The pharmaceutical compositions of the present invention are administered to a patient in need of such treatment by oral, nasal inhalation, rectal or parenteral administration. For oral administration, it can be made into conventional solid preparations such as tablet, powder, granule, capsule, etc., liquid preparations such as aqueous or oil suspension, or other liquid preparations such as syrup, elixir, etc.; for parenteral administration, it can be formulated into solution for injection, aqueous or oily suspension, etc.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient may be combined with one or more carriers and then formulated into the desired dosage form.

The embodiment of the invention has the following advantages:

the relative expression amount of genes of IL-6, IL-8, TNF- α, COX-2, Bax, Caspase 3 and ASCT2 in the pig intestinal epithelial cells induced by the DON can be remarkably reduced, and the expressions of genes ASCT2, ZO-1, Ocplus, Claudin-1, PePT1 and GLUT2 can be remarkably up-regulated.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a graph of the effect of different concentrations of DON and time on the viability of IPEC-J2 cells, wherein the different lower case letters are labeled to indicate significant difference (P <0.05) and the same letters indicate insignificant difference (P > 0.05);

FIG. 2 is a graph of the reduction of DON-induced cytotoxicity of CGA provided in accordance with an embodiment of the present invention, wherein (A) the reduction of DON-induced cytotoxicity of CGA at various concentrations; (B) the influence of different treatment groups on the LDH release rate is marked by different lower case letters to indicate that the difference is obvious (P <0.05), and the same letter indicates that the difference is not obvious (P > 0.05);

FIG. 3 is a graph of the relief of DON-induced apoptosis by CGA as provided in an embodiment of the present invention, wherein (A) flow cytometry analysis of Annexin V/FITC/PI stained apoptotic cells, with red, green, blue and pink representing late apoptotic cell rate (Q1), necrotic cell rate (Q2), viable cell rate (Q3) and early apoptotic cell rate (Q4), respectively; (B) statistical analysis of late apoptotic cell rate, necrotic cell rate, surviving cell rate, and early apoptotic cell rate. Labeling different lower case letters indicates significant difference (P <0.05), the same letter indicates insignificant difference (P > 0.05);

FIG. 4 is a graph of the effect of CGA on DON-induced cellular inflammatory gene relative mRNA abundance in accordance with an embodiment of the present invention, wherein the different lower case letters are labeled to indicate significant difference (P <0.05) and the same letter indicates insignificant difference (P > 0.05).

FIG. 5 is a graph of the effect of CGA on DON-induced apoptosis gene relative mRNA expression abundance in accordance with an embodiment of the present invention, wherein the different lower case letters are labeled to indicate significant difference (P <0.05) and the same letter indicates insignificant difference (P > 0.05).

FIG. 6 is a graph of the effect of CGA on DON-induced abundance of claudin gene versus mRNA expression, wherein the different lower case letters are labeled to indicate significant difference (P <0.05) and the same letter indicates insignificant difference (P > 0.05);

FIG. 7 is a graph of the effect of CGA on DON-induced abundance of nutrient transporter relative mRNA expression according to an embodiment of the invention, wherein the different lower case letters are labeled to indicate significant difference (P <0.05) and the same letter indicates insignificant difference (P > 0.05).

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. 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.

In the examples of the present invention, the test materials: CGA purity > 98% (HPLC), Phosphate Buffered Saline (PBS), 0.25% pancreatin-ethylenediaminetetraacetic acid (EDTA), penicillin-streptomycin, dimethyl sulfoxide (DMSO) and thiazolyl tetrazolium bromide (MTT) were purchased from Beijing Solibao Bio Inc.; DON purity > 99% was purchased from Sigma-Aldrich (USA); high glucose medium (HGDMEM) and Fetal Bovine Serum (FBS) were purchased from Biological Industries, Inc. (Israel); lactate Dehydrogenase (LDH) assay kit was purchased from tokyo bio-engineering institute of tokyo, south beijing, china.

In the embodiment of the invention, the IPEC-J2 cell line is provided by animal science and technology college of Jiangxi agricultural university. IPEC-J2 cell culture medium is HGDMEM medium containing 10% FBS and 1% penicillin-streptomycin, and is inoculated to 25cm²In a culture flask, and placing at 37 deg.C and 5% CO₂Cultured in an incubator. DON was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1mg/mL stock solution, which was then filtered through a 0.22 μm filter. CGA and DON are diluted into different concentrations by culture solution without serum and antibiotics, and are prepared for use.

In the following examples, six replicates of each treatment were run in the experiment, all data are presented as mean ± standard deviation, and all plots were generated using GraphPad Prism 7. All data were statistically analyzed by analysis of single variance (ANOVA) using the SPSS20.0 general linear model and compared multiple times using the Duncan test. The difference is significant when P <0.05, and the difference is not significant when P > 0.05.